Methods for detecting traumatic brain injury

ABSTRACT

The present invention provides detection reagents and method for determining risk of traumatic brain injury (TBI), assessment of the amount of neuronal damage, and/or susceptibility to neurodegenerative disease in a subject.

PRIORITY OF INVENTION

This application claims priority to U.S. Provisional Application No.62/141,003 that was filed on Mar. 31, 2015. The entire content of thisprovisional application is hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under W81XWH-12-1-0583and W81XWH-14-1-0467 awarded by the ARMY/MRMC. The government hascertain rights in the invention.

BACKGROUND

Traumatic Brain Injury (TBI) affects around 2 million people each year.This is likely an underestimate as many people with mild TBI may notseek medical care. Chronic stress and especially traumatic brain injury(TBI) disrupt cognitive function. Even mild traumatic brain injury(mTBI) frequently leads to chronic traumatic encephalopathy (CTE) and anincreased frequency and earlier onset of other brain disorders includingAlzheimer's disease (AD), Parkinson's disease (PD), frontotemporaldementia (FTD) and amyotrophic lateral sclerosis (ALS), so potentiallong term consequences of TBI can be quite devastating. There currentlyare no reliable methods to determine which TBI cases will developneurodegenerative disorders, and mechanisms by which TBI leads toneurodegeneration are poorly understood. Biomarkers of chronic TBI havenot been systematically studied and identification of biomarkers thatcan help identify which patients incurring TBI are most susceptible toneurodegenerative diseases such as AD would be extremely valuableclinical tools to help determine the appropriate course of care forveterans and others that have suffered brain injuries. There arecurrently no reliable physical diagnostic tests to determine traumaticbrain injury (TBI). Thus, methods and reagents are still needed to aidin the diagnosis of TBI.

SUMMARY

Methods have been developed that enable generation of reagents thatselectively bind disease related protein variants. The inventors havedeveloped methods and reagents to assess neuronal damage following TBI.Phage display antibody libraries are used as a source to isolate theprotein variant specific reagents. Also, a DARPin (Designed ankyrinrepeat protein) phage display library is used to generate and isolateadditional reagents selective for TBI. These libraries are used toidentify biomarkers that are indicative of different stages and severityof TBI.

In certain embodiments, the present invention provides a designedankyrin repeat protein (DARPin) comprising

(a) an N-Terminal Capping ankyrin repeat (AR) (SEQ ID NO:30),

(b) a C-Terminal Capping AR (SEQ ID NO:31), and

(c) three to six AR modules of about 30 to 35 amino acids, wherein eachAR module binds with a target.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at two or more timespost-injury;

(B) assessing protein levels of toxic variants of TDP-43, tau, abetaand/or alpha-synuclein in the sample by detecting protein levels oftoxic variants of TDP-43, tau, abeta and/or alpha-synuclein in thesamples;

(C) comparing the protein levels of toxic variants of TDP-43, tau, abetaand/or alpha-synuclein in the sample at each time point with proteinlevels of toxic variants of TDP-43, tau, abeta and/or alpha-synuclein ina normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having elevated protein levels of toxic variants ofTDP-43 tau, abeta and/or alpha-synuclein has a high risk of TBI.

As used herein, the term “toxic variant of TDP-43” is defined asvariants of TDP-43 that are preferentially present in diseased humantissue samples but not healthy control samples. Diseased tissue samplesinclude Alzheimer's disease, Parkinson's disease, FrontotemporalDementia, Lewy Body Dementia, Amyotrophic Lateral Sclerosis and otherneurodegenerative diseases.

As used herein, the term “toxic variant of tau” is defined as oligomerictau. In certain embodiments the oligomeric tau is dimeric tau, trimerictau, tetrameric tau, pentameric tau, hexameric tau, heptameric tau,octameric tau, nonameric tau, decameric tau, undecameric tau ordodecameric tau. In certain embodiments, the oligomeric tau is dimerictau or trimeric tau. In certain embodiments, the oligomeric tau istrimeric tau. (See, e.g., WO 2014/059442, which is incorporated byreference herein.)

As used herein, the term “toxic variant of abeta” is defined a smalloligomeric variant including trimeric and/or tetrameric forms of abeta.(See, e.g., WO 2012/058308, which is incorporated by reference herein.)

As used herein, the term “toxic variant of alpha-synuclein” is definedas oligomeric form of alpha-synuclein including dimeric, trimeric,tetrameric and higher order assemblies. (See, e.g., WO 2014/059442,which is incorporated by reference herein.)

In certain embodiments, a sample is obtained from the subject within 6hours post-injury.

In certain embodiments, a sample is obtained from the subject about 12to 36 hours post-injury.

In certain embodiments, a sample is obtained from the subject about 5 to10 days post injury.

In certain embodiments, a sample is obtained from the subject about 2 to4 weeks days post injury.

In certain embodiments, the sample and the normal control are bloodproduct samples or cerebrospinal fluid (CSF) samples. In certainembodiments, the blood product is serum.

In certain embodiments, the detecting in step (B) is by means of aligand specific for the protein.

In certain embodiments, the ligand is an antibody.

In certain embodiments, the ligand is a designed ankyrin repeat protein(DARPin).

In certain embodiments, the protein levels are detected by means ofELISA.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing levels of a toxic variant of TDP-43 in the sample bydetecting protein levels of a toxic variant of TDP-43 in the samples;

(C) comparing the protein levels of the toxic variant of TDP-43 in thesample at each time point with protein levels of the toxic variant ofTDP-43 in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having increased protein levels of the toxic variantof TDP-43 in the samples at all four time points than those of thenormal control, and having a decreased level of the toxic variant ofTDP-43 at the 24-hour time point as compared to the 6-hour time point,having a decreased level of TDP-43 at the 5-day time point as comparedto the 24-hour time point, and having an increased level of TDP-43 atthe 10-day time point as compared to either the 24-hour or 5-day timepoint has a high risk of TBI.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing levels of a toxic variant of tau in the sample bydetecting the protein levels of the toxic variant of tau in the samples;

(C) comparing the protein levels of the toxic variant of tau in thesample at each time point with protein levels of the toxic variant oftau in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having an increased protein levels of the toxicvariant of tau in the sample at the 24-hour time point and at the 5-daytime point as compared to that of the normal control, having comparableprotein levels of the toxic variant of tau at the 6-hour and 10-day timepoints as compared to that of the normal control, and having anincreased protein level of the toxic variant of tau at the 5-day timepoint as compared to the 24-hour time point has a high risk of TBI.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing protein levels of a toxic variant of abeta in the sampleby detecting protein levels of the toxic variant of abeta in thesamples;

(C) comparing protein levels of the toxic variant of abeta in the sampleat each time point with protein levels of the toxic variant of abeta ina normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having an increased protein levels of the toxicvariant of abeta in the sample at the 24-hour time point, the 5-day timepoint and the 10-day time point as compared to that of the normalcontrol, having comparable protein level of the toxic variant of abetaat the 6-hour as compared to that of the normal control, and having anincreased protein level of the toxic variant of abeta at the 5-day timepoint as compared to the 24-hour time point and 10-day time point has ahigh risk of TBI.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing protein levels of a toxic variant of alpha-synuclein inthe sample by detecting protein levels of the toxic variant ofalpha-synuclein in the samples;

(C) comparing the protein levels of the toxic variant of alpha-synucleinin the sample at each time point with protein levels of the toxicvariant of alpha-synuclein in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having an increased protein levels of the toxicvariant of alpha-synuclein in the sample at the 5 day time point, the10-day time point as compared to that of the normal control, havingcomparable protein level of the toxic variant of alpha-synuclein at the6-hour time point and 24-hour time point as compared to that of thenormal control, and having an increased protein level of the toxicvariant of alpha-synuclein at the 5-day time point as compared to the10-day time point has a high risk of TBI.

The present invention provides in certain embodiments a method formeasuring the presence of a biomarker in a human sample from a patienthaving traumatic brain injury (TBI), the improvement comprisingmeasuring the levels of toxic variants of TDP-43, tau, abeta and/oralpha-synuclein in the sample for use in predicting the amount ofneuronal damage, and/or susceptibility to neurodegenerative disease in asubject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. provides ELISA analysis of a DARPin clone with the TBI cases,which revealed reactivity at 24 hours, 5 days and 10 days, withreactivity decreasing after 5 days.

FIG. 2. provides details of the DARPin ALS biopanning procedures.

FIG. 3. provides highlights the procedures completed to obtain thepotential PD specific DARPins.

FIG. 4. provides highlights the portion of the schematic that summarizesthe AD biopanning procedures.

FIG. 5. Schematic of nanobody displayed on surface of phage particle.Multiple copies of pVIII coat protein enable over 1000-fold enhancementof nanobody signal.

FIG. 6. Oligomeric Aβ levels in human brain tissue from cognitivelynormal (ND), AD and PD samples. Combined oligomeric Aβ levels obtainedwith both A4 and C6T nanobodies are shown. The highest oligomeric Aβlevels are observed in AD samples with moderate plaques, then in ADsamples with severe plaques.

FIG. 7. Oligomeric a-syn levels in human brain tissue from cognitivelynormal (ND), AD and PD samples. Combined oligomeric a-syn levelsobtained with both D5 and 10H nanobodies are shown. High oligomerica-syn levels were present in PD samples but not AD or ND samples.

FIG. 8. Oligomeric tau levels in human brain tissue correlate with Braakstage. Oligomeric tau levels of brain homogenates were probed with theanti-oligomeric tau nanobody F9T. First bar: ND Braak stage I-II, noamyloid plaques. Second bar: ND Braak stage I-II slight plaques. Thirdbar: AD Braak stage III-IV moderate plaques. Fourth bar: AD Braak stageV-VI severe plaques. Oligomeric tau levels increase with Braak stage andthe presence of amyloid plaques.

FIG. 9. Oligomeric a-syn levels in post-mortem human CSF samples readilydistinguish PD from AD and ND cases. Combined oligomeric a-syn levelsobtained with both D5 and 10H nanobodies are shown. First bar: PDsamples, Second bar: AD samples, Third bar: ND samples High oligomerica-syn levels were present in PD samples but not AD or ND samples.

FIG. 10. Oligomeric Aβ levels in human sera from cognitively normal(ND), AD and PD samples. Combined reactivity with A4 and C6Tanti-oligomeric Aβ nanobodies are shown. First bar: ND samples with noplaques, Second bar: ND with moderate plaques. Third bar: AD withmoderate plaques. Fourth bar: AD with severe plaques. Fifth bar: PDsamples. Highest oligomeric Aβ levels are observed in AD samples withmoderate plaques, then in AD samples with severe plaques.

FIG. 11. Oligomeric a-syn levels in human sera from cognitively normal(ND), AD and PD samples. Combined oligomeric a-syn levels obtained withboth D5 and 10H nanobodies are shown. First bar: PD samples with lowp129 a-syn reactivity. Second bar: PD with moderate p129 a-synreactivity; Third bar: PD with high p129 a-syn reactivity. Fourth bar:AD samples. Fifth bar: ND. Highest oligomeric a-syn levels are observedin early stage PD samples with low levels in late stage PD, AD and NDsamples.

FIG. 12. An AD related TDP-43 variant is present in post-mortem humansera samples of AD but not cognitively normal (ND) samples. Samples wereanalyzed with a nanobody that selectively recognizes a TDP-43 variantpresent in AD and ALS but not ND brain tissue.

FIG. 13. Longitudinal plasma samples of AD (AD1-4) and control patients(C1-2) show presence of oligomeric Aβ in samples from all four patientsthat eventually converted to AD but not in control patients. Cumulativelevels of A4 and E1 reactive oligomeric Aβ variants are shown. Patientages are shown on x-axis. Samples taken after initial diagnosis of MCIare indicated by light shading and after diagnosis of AD by darkshading. Levels of oligomeric Aβ are detectable in all four patientsthat converted to AD but not in control patients. Significant oligomericAβ levels are present in AD samples well before conversion to AD, evenup to 7 years before initial diagnosis of MCI.

FIG. 14. Time course analysis of neurodegenerative disease relatedvariants of tau, Aβ, a-syn and TDP-43 in composite sera sample ofpatients after suffering acute severe traumatic brain injury. The signalobtained with each different protein variant is expressed relative tothe signal obtained with the control sera samples. Samples of 2 agedcontrol and 5 TBI cases were combined for analysis. TBI samples weretaken 6 hrs, 24 hrs, 5 days and 10 days after injury. Protein variantlevels are shown in different color bars: Tau (teal), Aβ (purple),a-syn, (orange) and TDP-43 (red). The tau, Aβ, a-syn and TDP-43 variantsare all present in the TBI composite sample, and all show different timedependencies following severe acute brain injury.

FIG. 15. Time course analysis of neurodegenerative disease relatedvariants of tau, Aβ, a-syn and TDP-43 in sera samples of five differentpatients after suffering acute severe traumatic brain injury. The signalobtained with each different protein variant is expressed relative tothe signal obtained with two aged control sera samples. TBI samples weretaken 6 hrs, 24 hrs, 5 days and 10 days after injury. Protein variantlevels are shown in different color bars: Tau (teal), Aβ (purple),a-syn, (orange) and TDP-43 (red). Patients P1 and P2 have primarily tauand a-syn toxic variants, P3 and P4 primarily toxic Aβ variants, and P5does not display significant levels of any toxic protein variants. Theseresults indicate that patients P1 and P2 have neuronal pathologymediated by tau and a-syn consistent with a risk of tauopathies andsynucleinopathies such as FTD and PD while patients P3 and P4 haveneuronal pathology mediated by Aβ consistent with a risk of AD.

FIG. 16. 3A Reactive TDP-43 Variant in Human TBI Sera.

FIG. 17. 3C Reactive TDP-43 Variant in Human TBI Sera.

FIG. 18. 8D Reactive TDP-43 Variant in Human TBI Sera.

FIG. 19. Cumulative TDP-43 Variants in Human TBI Sera.

FIG. 20. D11C Reactive Oligomeric Tau Levels in Human TBI Sera.

FIG. 21. C6T Reactive Oligomeric Beta-Amyloid Levels in Human TBI Sera.

FIG. 22. D5 Reactive Oligomeric Alpha-Synuclein Levels in Human TBISera.

FIG. 23. Cumulative Protein Variants Levels in Human TBI Sera.

FIG. 24A. 3A Reactive TDP-43 Variant in Human TBI Sera. FIG. 24B. 3CReactive TDP-43 Variant in Human TBI Sera. FIG. 24C. 8D Reactive TDP-43Variant in Human TBI Sera. FIG. 24D. Cumulative TDP-43 Variants in HumanTBI Sera.

FIG. 25A. D11C Reactive Oligomeric Tau in Human TBI Sera. FIG. 25B. C6TReactive Oligomeric Beta-Amyloid in Human TBI Sera. FIG. 25C. D5Reactive Oligomeric Alpha-Synuclein in Human TBI Sera. FIG. 25D.Cumulative Protein Variants in Human TBI Sera.

FIG. 26. Protein Variants in Human TBI Sera Samples (Outliers Present).

FIG. 27. Protein Variants in Human TBI Sera Samples (Outliers Removed).

FIG. 28. Protein Variants in TBI Sera (Outliers Removed).

FIG. 29. 3A Reactive TDP-43 Variant in Human TBI CSF.

FIG. 30. 3C Reactive TDP-43 Variant in Human TBI CSF.

FIG. 31. 8D Reactive TDP-43 Variant in Human TBI CSF.

FIG. 32. Cumulative TDP-43 Variants in Human TBI CSF.

FIG. 33. D11C Reactive Oligomeric Tau Levels in Human TBI CSF.

FIG. 34. A4 Reactive Oligomeric Beta-Amyloid Levels in Human TBI CSF.

FIG. 35. C6T Reactive Oligomeric Beta-Amyloid Levels in Human TBI CSF.

FIG. 36. 10H Reactive Oligomeric Alpha-Synuclein Levels in Human TBICSF.

FIG. 37. D5 Reactive Oligomeric Alpha-Synuclein Levels in Human TBI CSF.

FIG. 38. Cumulative Protein Variants Levels in Human TBI CSF.

FIG. 39A. 3A Reactive TDP-43 Variant in Human TBI CSF. FIG. 39B. 3CReactive TDP-43 Variant in Human TBI CSF. FIG. 39C. 8D Reactive TDP-43Variant in Human TBI CSF. FIG. 39D. Cumulative TDP-43 Variants in HumanTBI CSF.

FIG. 40A. D11C Reactive Oligomeric Tau in Human TBI CSF. FIG. 40B. A4Reactive Oligomeric Beta-Amyloid in Human TBI CSF. FIG. 40C. C6TReactive Oligomeric Beta-Amyloid in Human TBI CSF.

FIG. 41A. 10H Reactive Oligomeric Alpha-Synuclein in Human TBI CSF. FIG.41B. D5 Reactive Oligomeric Alpha-Synuclein in Human TBI CSF. FIG. 41C.Cumulative Protein Variants in Human TBI CSF.

FIG. 42. Protein Variants in Human TBI CSF Samples (Outliers Present).

FIG. 43. Protein Variants in Human TBI CSF Samples (Outliers Removed).

FIG. 44. Protein Variants in TBI CSF.

FIG. 45A. Line Graph Comparison of 3A Reactive TDP-43 Variant in HumanTBI Sera and CSF. FIG. 45B. Bar Graph Comparison of 3A Reactive TDP-43Variant in Human TBI Sera and CSF. FIG. 45C. Line Graph Comparison of 3CReactive TDP-43 Variant in Human TBI Sera and CSF. FIG. 45D. Bar GraphComparison of 3C Reactive TDP-43 Variant in Human TBI Sera and CSF.

FIG. 46A. Line Graph Comparison of 8D Reactive TDP-43 Variant in HumanTBI Sera and CSF. FIG. 46B. Bar Graph Comparison of 8D Reactive TDP-43Variant in Human TBI Sera and CSF. FIG. 46C. Line Graph Comparison ofD11C Reactive Oligomeric Tau in Human TBI Sera and CSF. FIG. 46D. BarGraph Comparison of D11C Reactive Oligomeric Tau in Human TBI Sera andCSF.

FIG. 47A. Line Graph Comparison of C6T Reactive Oligomeric Beta-Amyloidin Human TBI Sera and CSF. FIG. 47B. Bar Graph Comparison of C6TReactive Oligomeric Beta-Amyloid in Human TBI Sera and CSF. FIG. 47C.Line Graph Comparison of A4 Reactive Oligomeric Beta-Amyloid in HumanTBI Sera and CSF. FIG. 47D. Bar Graph Comparison of A4 ReactiveOligomeric Beta-Amyloid in Human TBI Sera and CSF.

FIG. 48A. Line Graph Comparison of D5 Reactive OligomericAlpha-Synuclein in Human TBI Sera and CSF. FIG. 48B. Bar GraphComparison of D5 Reactive Oligomeric Alpha-Synuclein in Human TBI Seraand CSF. FIG. 48C. Line Graph Comparison of 10H Reactive OligomericAlpha-Synuclein in Human TBI Sera and CSF. FIG. 48D. Bar GraphComparison of 10H Reactive Oligomeric Alpha-Synuclein in Human TBI Seraand CSF.

DETAILED DESCRIPTION

In order to determine which patients suffering TBI are at high risk ofincurring AD, it is necessary to understand the pathology and riskfactors of AD. The primary constituents of the two major pathologicalfeatures of AD, neurofibrillary tangles and amyloid plaques, arerespectively, the tau and Aβ proteins. The two most promising biomarkersfor AD to date are variants of tau and Aβ, in particular phosphorylatedvariants of tau and the 42 amino acid variant of Aβ (A(342). While tauand Aβ biomarkers suffer from a relatively low sensitivity andspecificity for diagnosing AD, they still hold great promise for earlydetection of AD as changes in CSF levels of tau and Aβ42 have been shownto occur well before symptoms develop, up to 25 years earlier for Aβ42.While CSF tau and Aβ levels correlate with AD, the vast majority ofstudies have focused on detection of non-toxic monomeric forms of Aβ42and phosphorylated tau rather than on detection of the actual toxicprotein species responsible for neurodegeneration. Both tau and Aβ canexist in a variety of different forms and aggregate morphologies andnumerous studies indicate that specific oligomeric forms of both tau andAβ are involved in neuron degeneration and spread of toxicity, and caninterfere with important functions such as long term potentiation.Therefore a potential route for presymptomatic diagnosis of AD which canalso help to identify those at high-risk of incurring AD is tospecifically detect the individual protein species that are involved invery early stages of disease onset and progression. Because misfoldedand aggregated variants of tau and Aβ are intimately involved in theprogression of AD, detection of specific variants of these proteins inCSF and especially serum has great promise for an early definitivediagnosis of AD. In a parallel manner, misfolded toxic oligomericvariants of the protein a-syn have been correlated with the onset andprogression of PD and related synucleinopathies and aggregated toxicvariants of Tar DNA binding protein 43 (TDP-43) have been correlatedwith ALS, FTD and AD. Therefore the presence of selected toxic variantsof tau, Aβ, TDP-43 and a-syn all have potential value as early andsensitive diagnostic biomarkers for different neurodegenerative diseasesand also to identify individuals at high risk of incurring thesediseases.

The brain is very sensitive to stress and injury and responds byexpressing a variety of neuromorphological and neurochemical changes.Injury to the brain can alter neuronal protein function through avariety of different ways. Cellular stress can affect processing ofproteins by altering genetic splicing, protein production andpost-translational modification, it can alter how RNA binding proteinsinteract with mRNA affecting protein translation, and it can also alterprotein folding and aggregation states. All of these changes can resultin formation of toxic protein variants that result in a loss of neuronalfunction and potentially cell death. Some of the key biochemical changesobserved in the brain following TBI are similar to changes seen inpatients suffering from neurodegenerative diseases including AD, PD, ALSand FTD. As one example, stress induces increased neuronal expression oftwo proteins implicated in AD, the Amyloid Precursor Protein (APP) andBACE-1 a protease which cleaves APP to generate the amyloid-beta (Aβ)protein. Increase in expression levels of APP and BACE-1 can lead to anincrease in Aβ deposition, similar to what is seen in AD patients, animportant feature since patients suffering brain trauma are at greaterrisk of developing AD and at an earlier age. A second example ofbiochemical changes observed following TBI mirroring those that occur inneurodegenerative diseases are the morphological changes induced in theprotein tau. Neuronal axons are particularly vulnerable to the highsheer forces and mechanical deformation induced by TBI. Resulting damageto the neuronal axons can impair protein transport leading toaccumulation of proteins including tau and subsequent swelling causingthe typical axon pathology observed with TBI. Tau plays a critical rolein neuronal damage following TBI and increased levels of tau in brainfluid, CSF and serum samples are all predictive of adverse long-termclinical outcomes after TBI. Neurofibrillary tau aggregates have beenidentified in soldiers suffering from TBI and in athletes such asfootball players who suffer repeated head trauma. Aggregates of tau arealso the major component of the hallmark neurofibrillary tangles in ADbrain, and TBI is a risk factor for AD. Therefore TBI can directly leadto formation of Aβ and tau variants that are also associated with AD.

Injury and cellular stress can contribute to an increase in productionof other misfolded and aggregated proteins in addition to Aβ and tau,all of which can overwhelm cell clearance mechanisms affectingproteostasis eventually leading to neuronal degeneration. Similar to theroles of Aβ and tau in AD and other tauopathies, the proteinalpha-synuclein (a-syn) plays an important role in the onset andprogression of PD and other related neurodegenerative disorders as it isa major component of the hallmark Lewy body aggregates associated withPD. Specific variants of a-syn including aggregated and phosphorylatedvariants have been correlated with PD, so formation of toxic a-synvariants following TBI may also play a role in the increased incidenceof PD associated with brain trauma. Similarly cytoplasmic misfolding andaggregation of TDP-43 have been associated with ALS. TDP-43 binds to avariety of RNA and DNA sequences, particularly to poly-UG RNA sequencesaccounting for its location in the nucleus, but it can shuttle back andforth from the cytoplasm. In affected neurons and glial cells of ALScases a variety of different TDP-43 forms accumulate in inclusion bodiesin the cytoplasm and/or nucleus rather than in their normal diffusedistribution pattern in the nucleus. Following brain injury or stress,TDP-43 co-localizes with stress granules in the cytoplasm promotingformation of inclusion bodies containing aggregated TDP-43 variants.TDP-43 aggregates have also been correlated with FTD and AD pathology.Therefore brain injury and the neuronal stress and cellular changesresulting from the injury can lead to generation of a variety ofdifferent protein variants in particular toxic variants of tau, Aβ,a-syn and TDP-43 all of which have also been implicated in the onset andprogression of neurodegenerative diseases including AD, PD, FTD and ALS.

While misfolding of certain proteins has been associated with neuronaldamage and disease, more than one protein is likely to misfold andaggregate in brain tissue complicating diagnosis and treatmentstrategies. Since cellular stress induced by misfolding and aggregationof one protein may well lead to misfolding and aggregation of otherproteins, the presence of multiple misfolded proteins in differentdiseases should be expected. For example injury induced aggregation oftau can lead to misfolding, aggregation or altered processing of Aβ anda-syn, complicating diagnosis of a specific neurodegenerative disease.Increased levels of Aβ variants are known to increase both tau andTDP-43 pathology indicating a link between key aggregation proneneuronal proteins including Aβ, tau, a-syn, and TDP-43. Therefore, thereis likely a spectrum of diseases that can be caused by brain injuryinvolving various different toxic protein variants targeting differentcells and regions. Since this spectrum of neurodegenerative diseasesshare overlapping features, carefully characterizing the differentprotein variants that are present in patient serum samples and comparingthese to the protein variant profiles that are characteristic ofdifferent neurodegenerative disease would greatly facilitate earlydiagnosis of the appropriate neurodegenerative disease and also help toidentify and monitor the most appropriate treatment strategies. Thepresence of select key disease related variants of tau, Aβ, a-syn, andTDP-43 in serum following TBI therefore represents a very powerfuldiagnostic tool to assess the type and extent of neuronal damage inducedby the injury and can be useful to predict which patients are mostsusceptible to particular neurodegenerative diseases. While a number ofCSF based biomarkers including different monomeric forms of Aβ, tau anda-syn have shown promise for diagnosing some neurodegenerative diseases,early and accurate diagnosis of different neurodegenerative diseases isstill not feasible. Detection and quantification of selected toxicprotein variants that are associated with the onset and progression ofAD and other neurodegenerative diseases have great promise as tools tofacilitate early diagnosis and to identify patients who are at high riskof neurodegenerative disease. Current biomarker studies of TBI patientshave not been particularly successful as S100B, a calcium bindingprotein has been the only marker to consistently predict TBI andoutcome. Since S100B has also been implicated in various other diseasesincluding diabetes, melanoma and epilepsy, its use in predicting TBI islimited, and supplemental information regarding toxic protein variantswould be very beneficial. This proposal seeks to develop appropriatebiomarkers to identify which patients are at risk of AD and otherneurodegenerative diseases and to determine the extent of neuronaldamage following TBI.

In order to determine the most promising biomarkers to identifyindividuals at high risk of developing specific neurodegenerativediseases such as AD, it was first necessary need to identify the mostrelevant protein variants associated with each neurodegenerativedisease. While the fibrillar forms of both tau and Aβ are respectivelyfound in the neurofibrillary tangles and amyloid plaques characteristicof AD, these aggregates are not particularly neurotoxic and do notcorrelate well with disease onset and severity. Small soluble oligomericforms of both proteins however do correlate much better with diseaseoutcomes and different oligomeric forms are thought to be responsiblefor spread of pathology in the brain. Oligomeric forms of both Aβ andtau are acutely neurotoxic and are key features in the neurodegenerativephenotype. Oligomeric Aβ and tau species have been shown to contributeto neurotoxicity through an “infectious” model of disease progression.Extracellular tau aggregates can initiate tau misfoldingintracellularly, tau pathology spreads contiguously throughout the brainfrom early to late stage disease, and brain extract from a transgenicmouse with aggregated mutant human tau transmits tau pathology whenintroduced into the brains of mice expressing normal human tau.Therefore oligomeric forms of both Aβ and tau are promising earlybiomarkers to track neuronal damage following TBI.

In a parallel manner to the roles of oligomeric Aβ and tau in AD,numerous studies also indicate that oligomeric aggregates of a-syn causeneuronal toxicity and induce spread of pathology in PD. Differentoligomeric variants of a-syn were shown to be toxic to dopaminergicneurons in vivo and in cell models. We have shown that oligomeric butnot fibrillar forms of a-syn are toxic to neuronal cells. Toxicoligomeric a-syn forms were identified in living cells and in humanplasma from PD patients. Therefore oligomeric a-syn variants are alsopromising early biomarkers for neuronal damage following TBI that may beindicative of PD and other synucleinopathies. Detection of specifictoxic protein variants of Aβ, a-syn or tau has shown promise fordiagnosis of AD and PD providing strong precedent that identifying thetoxic protein variant fingerprint associated with differentneurodegenerative diseases has excellent potential to be a powerful toolto facilitate early diagnosis of specific neurodegenerative diseases andto identify patients at risk of incurring these diseases. While proteinvariants of Aβ, a-syn and tau have demonstrated value in diagnosingdifferent neurodegenerative diseases, the presence of different variantsof a fourth protein, TDP-43, also has promise to sharpen the diagnosticcapabilities of the protein variants because of its important role inALS, FTD and AD. TDP-43 accumulation occurs in different brain regionsin ALS and in different types of FTD, suggesting that TDP-43 aggregationcan be a useful diagnostic biomarker for these diseases. Presence ofTDP-43 inclusions is also evident in a subset of AD cases, primarily inlimbic regions where it can overlap with tau pathology. FTD-43 pathologycan be induced by increased expression of Aβ providing additionalevidence of the link between key aggregation prone proteins in the brainincluding Aβ, tau, a-syn, and TDP-43. Since tau, Aβ, a-syn, and TDP-43all play key roles in brain function and neurodegeneration, following isa brief discussion of how each protein contributes to neuronal functionand the different protein variants that lead to neurodegeneration.

Tau. The microtubule associating protein tau is a major component of theneurofibrillary tangles associated with AD and tauopathies that arecharacterized by hyperphosphorylation and aggregation of tau. Tau playsan important role in assembly and stabilization of microtubules. Tau isa natively unfolded protein, and similar to a number of other nativelyunfolded proteins, it can aberrantly fold into various aggregatemorphologies including β-sheet rich fibrillar forms. The different typesof post-translational modifications of tau in AD includephosphorylation, glycosylation, glycation, prolyl-isomerization,cleavage or truncation, nitration, polyamination, ubiquitination,sumoylation, oxidation and aggregation. Tau has 85 putativephosphorylation sites, and excess phosphorylation can interfere withmicrotubule assembly. Tau can be modified by phosphorylation or byreactive nitrogen and oxygen species among others. Tau is anintrinsically unstructured protein due to its very low hydrophobiccontent containing a projection domain, a basic proline-rich region, andan assembly domain. Hexapeptide motifs in repeat regions of tau give theprotein a propensity to form β-sheet structures which facilitateinteraction with tubulin to form microtubules as well asself-interaction to form pathological aggregates such as paired helicalfilaments (PHF). Hyperphosphorylation of tau, particularly in theassembly domain, decreases the affinity of tau to the microtubules andimpairs its ability to regulate microtubule dynamics and axonaltransport. In addition, parts of the basic proline-rich domain and thepseudo-repeat also stabilize microtubules by interacting with itsnegatively charged surface. Alternative splicing of the second, thirdand tenth exons of tau result in formation of six tau isoforms. Theassembly domain in the carboxyl-terminal portion of the protein containseither three or four repeats (3R or 4R) of a conserved tubulin-bindingmotif depending on alternative splicing of exon 10. Tau 4R isoforms havegreater microtubule binding and stabilizing ability than the 3Risoforms. Human adult brains have similar levels of 3R and 4R isoforms,while only 3R tau is expressed at the fetal stage. Mutations alteringsplicing of tau transcript and the ratio of 3R to 4R tau isoforms aresufficient to cause neurodegenerative disease. Therefore tau in humanbrain tissue can exist in a variety of different lengths andmorphologies and with multiple post-translational modifications.

Tau plays a critical role in the pathogenesis of AD and studies showthat reduction of tau levels in AD animal models reverses diseasephenotypes and that tau is necessary for the development of cognitivedeficits in AD models caused by over-expression of Aβ. Whileneurofibrillary tangles (NFTs) have been implicated in mediatingneurodegeneration in AD and tauopathies, animal models of tauopathy haveshown that memory impairment and neuron loss do not associate well withaccumulation of NFT. Animal studies showed improvement in memory andreduction in neuron loss despite the accumulation of NFTs, a regionaldissociation of neuron loss and NFT pathology, and hippocampal synapseloss and dysfunction and microglial activation months before theaccumulation of filamentous tau inclusions. The pathological structuresof tau most closely associated with AD progression are tau oligomers.All these studies suggest that tau tangles are not acutely neurotoxic,but rather that pretangle oligomeric tau species are responsible for theneurodegenerative phenotype, similar to toxic role of oligomeric Aβspecies.

Numerous studies suggest that extracellular tau species contribute toneurotoxicity through an “infectious” model of disease progression. Forexample, tau pathology spreads contiguously throughout the brain fromearly to late stage disease, extracellular tau aggregates can propagatetau misfolding from the outside to the inside of a cell, brain extractfrom a transgenic mouse with aggregated mutant human tau transmits taupathology throughout the brain in mice expressing normal human tau,induction of pro-aggregation human tau induces formation of tauaggregates and tangles composed of both human and normal murine tau(co-aggregation), and levels of tau rise in CSF in AD, whereas Aβ levelsdecrease. A receptor-mediated mechanism for the spread of tau pathologyby extracellular tau has been identified.

Elevated total tau concentration in CSF has been correlated with AD, ashas the presence of various phosphorylated tau forms, and the ratio oftau to Aβ42. Reactive nitrogen and oxygen can modify tau facilitatingformation of aggregate forms including oligomeric species. Levels ofoligomeric tau have also been implicated as a potential early diagnosticfor AD. Therefore, while determination of total tau and phosphorylatedtau levels has demonstrated value for diagnosis of AD and othertauopathies, reagents that can selectively recognize the tau speciesthat are most selectively involved in AD and TBI, especially thosepresent in sera samples, would have particular value in earlydiagnostics of neurodegenerative diseases including tauopathies and ADand assessing neuronal damage and risk of AD in TBI patients.

Aβ. The principle component of the extracellular neuritic plaquesimplicated in AD is the β-amyloid protein (Aβ), an approximately 4 kDafragment proteolytically derived from the larger amyloid precursorprotein (APP). A vast amount of literature has implicated Aβaccumulation as being central to the progression of AD, leading toformation of the Aβ hypothesis postulating a central role for Aβ in AD.Mutations or polymorphisms in four genes, APP, Presenilins 1 and 2, andApolipoprotein E4 were correlated with AD and each of these genes wasconnected with an increased production or decreased clearance of Aβ,particularly Aβ42. An increased gene dosage of APP resulting fromtrisomy 21 of Down Syndrome also leads to increased presence of neuriticplaques and AD. Numerous in vitro studies demonstrated the cytotoxicityof aggregated Aβ samples, and defined various conditions that favoraggregation. Despite all the evidence suggesting a strong role of Aβ inthe progression of AD, the major weakness of the Aβ hypothesis is thatthe presence of amyloid plaques does not correlate well with theprogression of AD, and considerable controversy exists over the role andmechanism of Aβ in AD. Deterioration of synapse integrity, particularlydendritic arbors, correlates well with dementia, however in animalmodels of AD fibrillar amyloid deposits were not necessary for synapseloss, and in immunized mice memory loss could be reversed without theremoval of amyloid plaques. Small soluble oligomeric Aβ aggregates wereshown to be potent neurotoxins suggesting that other non-amyloidaggregate forms of Aβ may be the relevant toxic species in AD, leadingto a revised “oligomer Aβ hypothesis”.

Many studies have confirmed the role of oligomeric Aβ in AD pathology,although not without considerable confusion. Cortical levels of solubleAβ correlate well with cognitive impairment and loss of synapticfunction. Small, soluble aggregates of Aβ termed Aβ-derived diffusibleligands (ADDLs) and spherical or annular aggregates termed protofibrilswere shown to be neurotoxic. Oligomeric forms of Aβ, created in vitro orderived from cell cultures, were shown to inhibit long term potentiation(LTP). Early stage memory loss associated with inhibition of LTP couldbe reversed by administering anti-Aβ antibodies to transgenic mousemodels of AD. The concentration of oligomeric forms of Aβ are alsoelevated in transgenic mouse models of AD and in AD brain. Disruption ofneural connections was shown to occur near Aβ plaques and fibrils,suggesting a toxic role for the fibrillar form, although the disruptionalso occurred in regions without fibrillar Aβ deposition suggesting thatthe toxicity may be due to small amounts of oligomeric Aβ, some inequilibrium with the fibrillar form, some existing on their own. A haloof oligomeric Aβ was shown to surround Aβ plaques and correlate withsynapse loss. Oligomeric Aβ was also shown to disrupt cognitive functionin transgenic animal models of AD. These selected studies, along withmany others as a whole, suggest that oligomeric Aβ is involved insynapse failure and early memory loss in AD. In addition, levels ofsmall soluble Aβ aggregates in CSF were shown to correlate with advancedAD cases. Therefore there are numerous studies indicating that varioussoluble aggregated Aβ species are intimately involved in theneurological decline associated with AD.

A-syn. PD is the second most prevalent neurodegenerative diseasefollowing AD, affecting around 2% of the people over the age of 65,although the disease progresses over many years. PD results indisturbances in motor function characterized by tremor, rigidity andbradykinesia. A 50 to 70% loss of dopaminergic neurons in the substantianigra and loss in other regions of the nervous system and the presenceof Lewy bodies and Lewy neurites are all indicative of PD. Lewy bodiesare intracellular protein inclusions composed of a dense core offilamentous and granular material coated with radially orientedfilaments. Lewy neurites contain filaments that are structurally andimmunologically similar to those found in Lewy bodies. Lewy body andneurites are present in both peripheral and central neurons in PD andseem to progress in a caudal to rostral fashion; they are likelyassociated either with neuronal dysfunction or with neuronal deathdepending on the brain region and the stage of the disease. While mostcases of PD are sporadic, or non-familial linked, there are a smallpercentage of cases that are genetically inherited. From these earlyonset familial PD cases, several mutations in the a-syn gene have beencorrelated with PD. The a-syn protein is expressed rather abundantly inbrain cells, localizing in the presynaptic terminal and is a majorcomponent of Lewy bodies and neurites. Expression levels of a-syncontribute to PD as increased gene expression of wild type a-syn wascorrelated with early onset cases of PD. Genome Wide Association studieshave also confirmed the association of a-syn with sporadic PD. Two othergenetic factors that correlate with PD, Parkin, and ubiquitin C-terminalhydrolase L1, are involved in the ubiquitin-proteasome pathway, aclearance system that fails in PD, resulting in the accumulation ofheavily ubiquitinated a-syn aggregates. There is considerablecontroversy over the role that clearance of a-syn plays in PD. Studiesindicate that a-syn can be cleared from cells by either theubiquitin-proteosome system or by the autophagy-lysosomal pathway. Thetwo clearance systems may work in concert with each other as inhibitingclearance of a-syn by one system increases clearance through the othersystem. A-syn contains a chaperone mediated autophagy sequence that mayfacilitate translocation across the lysosomal membrane throughinteraction with the heat shock cognate protein 70 facilitating itsdegradation. Blocking this clearance mechanism leads to accumulation ofinsoluble oligomeric a-syn. Mutant forms of a-syn including A30P andA53T that are more prone to aggregation bind more readily to thelysosomal membrane, but are less efficiently internalized providingfurther evidence that the aggregation state of a-syn is a criticalfactor in its function and toxicity. The aggregation state of a-syn hasbeen strongly correlated with synucleinopathies including PD and LBD.Aggregated forms of a-syn induce toxicity in dopaminergic neurons invivo, several different oligomeric morphologies were shown to havedifferent toxic mechanisms and we have shown among others thatoligomeric but not fibrillar forms of the protein are neurotoxic. Toxicoligomeric a-syn forms were identified in living cells and in humanplasma and CSF from human PD patients. We showed that intracellulartargeting and clearance of oligomeric a-syn completely protectedmammalian cells against toxicity induced by a-syn overexpression.Oligomeric but not monomeric forms of a-syn induce fragmentation ofmitochondria. Monomeric and oligomeric a-syn can also interfere withmitochondrial function by interacting with the endoplasmic reticulum(ER) and inducing stress. Increased levels of a-syn and in particularoligomeric a-syn species can increase Ca+2 influx into cells, which canthen promote further aggregation of a-syn. Oligomeric species of a-synalso have been shown to affect neurotransmitters, disrupting theglutamatergic system. Clearly different protein variants of a-syn playkey roles in the onset and progression of PD and therefore have greatpromise as early biomarkers for PD and other synucleinopathies.

TDP-43. Increasing evidence indicates that aggregates of TDP-43 play arole in FTD, ALS and other neurodegenerative diseases includingtraumatic brain injury. Similar to other neuronal proteins including Aβ,a-syn and tau, TDP-43 is prone to form aggregate species, where TDP-43mutations linked to increased risk of sporadic ALS aggregate morereadily. Initial studies implicated superoxide dismutase (SOD-1) in ALS,however only a small percentage of ALS cases are linked to mutations inSOD-1 whereas cytoplasmic aggregates of TDP-43, a protein normallyprimarily located in the nucleus, are found in a vast majority ofsporadic ALS cases. TDP-43 is a DNA binding protein which has a numberof different alternatively spliced forms. TDP-43 binds to a variety ofRNA and DNA sequences, particularly to poly-UG RNA sequences accountingfor its location in the nucleus, but it can shuttle back and forth fromthe cytoplasm. In FTD and ALS cases, affected neurons and glial cellsshow similar pathology where a variety of different TDP-43 formsaccumulate in inclusion bodies in the cytoplasm and/or nucleus with lossof the normal diffuse nuclear distribution.TDP-43 accumulation occurs indifferent regions with different types of FTD, suggesting that TDP-43aggregation can be a useful diagnostic biomarker for these diseases.Presence of TDP-43 inclusions is also evident in a subset of AD cases,primarily in limbic regions where it can overlap with tau pathology.TDP-43 co-localizes with stress granules in the cytoplasm followingstress and it contains a prion like domain which may account for theability of TDP-43 pathology to spread from diseased to healthy cells.Increased CSF levels of TDP-43 have been observed in ALS patientsproviding evidence that toxic TDP variants can spread from cell to cell.TDP-43 aggregates have also been correlated with FTD and AD pathology.Aggregation of TDP-43 in cell cytoplasm is considered to induce toxicityby both a toxic gain of function of the TDP-43 aggregates and a loss ofbeneficial function of soluble TDP-43 in the nucleus. Stress and agingare both factors that contribute to an increase in production ofmisfolded and aggregated proteins, which in turn can overwhelm cellclearance mechanisms affecting proteostasis eventually leading toneuronal degeneration. FTD-43 pathology can be induced by increasedexpression of Aβ providing additional evidence of the link between keyaggregation prone proteins in the brain including Aβ, tau, a-syn, andTDP-43. There is therefore substantial evidence that aggregation ofTDP-43 is an important factor in FTD, ALS and other neurodegenerativediseases, and detection of specific disease related TDP-43 variants alsohas great promise as early biomarkers for neurodegenerative diseasesincluding AD, ALS and FTD.

Protein misfolding and aggregation is clearly a critically importantfeature in neurodegenerative diseases, so determining how concentrationprofiles of selected key forms and morphologies of tau, Aβ, TDP-43 anda-syn vary in AD, TBI and cognitively normal patients facilitatesdevelopment of an effective diagnostic assay for these disorders.Different toxic oligomeric aggregates are generated in the brainfollowing TBI increasing the subsequent risk for a spectrum ofneurodegenerative disorders including AD, PD, FTD and other dementias.Accurate characterization of which specific protein variants oraggregates are present in tissue and serum samples at differenttimepoints following TBI provide very powerful biomarkers to bothproduce a more precise picture of the molecular processes that occur inthe brain as a function of time after brain injury and also tofacilitate diagnosis and direct appropriate therapeutic strategies foreach individual injury case.

Quantification of serum levels of the actual toxic protein variantsinvolved in onset and progression of key neurodegenerative diseasesprovide a much more sensitive and powerful set of biomarkers to assessneuronal damage following TBI and for early detection and staging ofresulting neurodegenerative diseases such as AD that result from TBI.While detection of these specific protein variants has great promise,such studies have not been feasible due to the low concentrations of thetarget protein variants in CSF and particularly serum samples and thepoor specificity of reagents for the different protein species. Toovercome this problem, novel technology was developed that combines theimaging capabilities of AFM with the binding diversity of phage displayantibody technology which enables allow us to identify the presence ofspecific protein variants and then isolate reagents that bind the targetvariant (Barkhordarian, H., et al., Protein Eng Des Sel, 2006. 19(11):p. 497-502). Utilizing this technology antibody based (nanobody)reagents were generated that very selectively recognize different toxicvariants of tau, Aβ, TDP-43 and a-syn (Emadi, S., et al., J Mol Biol,2007. 368(4): p. 1132-44; Barkhordarian, H., et al., Protein Eng DesSel, 2006; Emadi, S., et al., J Biol Chem, 2009. 284(17): p. 11048-58;Emadi, S., et al., Biochemistry, 2004. 43(10): p. 2871-2878; Zhou, C.,et al., Mol Ther, 2004. 10(6): p. 1023-31; Liu, R., et al.,Biochemistry, 2004. 43(22): p. 6959-67; Zameer, A., et al., J Mol Biol,2008. 384(4): p. 917-28; Zameer, A., et al., Biochemistry, 2006. 45(38):p. 11532-9; Kasturirangan, S., et al., Neurobiol Aging, 2012. 33(7): p.1320-8) Also, a simple novel sandwich ELISA was developed that enablesfemtomolar or better detection of specific target antigens directly frombiological samples including CSF and serum samples. Here a panel ofnanobody reagents against variants of tau, Aβ, TDP-43 and a-syn areutilized to demonstrate that different specific protein variant speciesare generated following TBI depending on the extent and type of injury,and that detection and quantification of these different proteinvariants may identify patients at high risk of specificneurodegenerative diseases including AD.

There is an urgent need for biomarkers for TBI as S100B he only markerto consistently predict TBI and outcome to date, however S100B has alsobeen implicated in other diseases including AD, diabetes, melanoma andepilepsy. The nanobodies we generated for use here selectively recognizetoxic protein variant biomarkers that are associated with specificneurodegenerative diseases, therefore these nanobodies are recognizingbiomarkers that are selectively associated with the onset andprogression of different types of neuronal damage rather thanrecognizing a more generic secondary effect such as inflammatorysignals, microglial activation or apoptotic markers. Three differentnanobodies against different oligomeric AP species have been shown toselectively distinguish between AD and PD or healthy samples inpost-mortem human tissue (Zameer, A., et al., J Mol Biol, 2008. 384(4):p. 917-28) and CSF samples (Sierks, M. R., et al., Integrative Biology,2011. 3(12): p. 1188-96). It has similarly been shown that two differentnanobodies against different toxic oligomeric a-syn species bothselectively distinguish between PD and AD or healthy post-mortem humantissue (Emadi, S., et al., J Mol Biol, 2007. 368(4): p. 1132-44; Emadi,S., et al., J Biol Chem, 2009. 284(17): p. 11048-58) and CSF samples(Sierks, M.R., et al., Integrative Biology, 2011. 3(12): p. 1188-96). Ithas also been shown that nanobodies against a toxic trimeric tau speciesdistinguish AD from healthy post-mortem human tissue, and that we cangenerate nanobodies against TDP-43 variants that distinguish ALS and FTDpost-mortem human tissue samples from healthy samples.

As described below a bank of well characterized serum samples from takenfrom patients show incurred different severities of TBI taken at variousacute (up to 24 hours after injury) and chronic (12-36 months) timepoints following injury was established. The results show that it waspossible to readily distinguish and stage progression ofneurodegenerative diseases when using particular nanobodies to assaypost-mortem tissue, CSF and serum samples. The results also show theability to readily distinguish longitudinal serum samples from patientssubsequently diagnosed with AD from aged matched cognitively normalpatients. The results readily detect oligomeric Aβ in sera samples ofpatients that converted to AD at least seven years prior to initialdiagnosis of mild-cognitive impairment (MCI) providing strong precedentfor a blood based presymptomatic diagnosis of AD. The results also showthat nanobodies against an AD variant of TDP-43 can also readilydistinguish AD serum samples from controls. Finally, the results showthe ability to detect the presence of neurodegenerative disease relatedvariants of tau, Aβ, a-syn and TDP-43 in serum samples from patientsthat have suffered severe acute TBI, and that different patients showdistinctly different protein variant fingerprints, some indicative ofAD, others indicative of PD or FTD. These results indicate thatdistinctly different cellular processes occur in individual patientsafter suffering TBI, the different cellular process leave distinctprotein variant fingerprints and that these fingerprints may indicateincreased risk for specific neurodegenerative diseases. Since all thenanobodies used here recognize protein variants that are found indifferent diseased but not healthy samples, these nanobodies have greatutility as presymptomatic biomarkers for different neurodegenerativediseases, and to identify individuals who are susceptible to ADfollowing TBI.

Designed Ankyrin Repeat Proteins

DARPins (designed ankyrin repeat proteins) are genetically engineerednon-immunoglobulin antibody-mimetic proteins that offer advantages overantibodies for target binding in drug discovery and drug development.DARPins have been successfully used, for example, for the inhibition ofkinases, proteases and drug-exporting membrane proteins. DARPinstypically exhibiting highly specific and high-affinity target proteinbinding. They are derived from natural ankyrin proteins and consist ofat least three, usually four or five repeat motifs of these proteins.Their molecular mass is about 14 or 18 kDa (kilodaltons) for four- orfive-repeat DARPins, respectively.

Naturally occurring ankyrin proteins form a class of proteins thatmediate high-affinity protein-protein interactions in nature. Severalthousand natural ankyrin repeat motifs (of about 33 amino acids each)are known, and can be combined with structure based design andrecombinant DNA methods for generation of novel proteins. Theserepetitive structural units form a stable DARPin protein domain with alarge potential target interaction surface. Typically, DARPins arecomposed of four or five repeats, corresponding to the average size ofnatural ankyrin repeat protein domains. Proteins with less than threerepeats do not form a tertiary structure. Libraries of DARPins withrandomized potential target interaction residues with diversities ofover 10¹² variants have been generated at the DNA level. From theselibraries, DARPins binding the target of choice with picomolar affinityand specificity can be selected using ribosome display or signalrecognition particle (SRP) phage display.

DARPins can be designed to act as receptor agonists, antagonists,inverse agonists, enzyme inhibitors, or simple target protein binders.The DARPins exhibit high thermal and thermodynamic stability(denaturation midpoint: Tm>66° C., equilibrium unfolding: ΔG>9.5kcal/mol), which increases with increasing repeat number. DARPins arestable in human blood serum and do not contain T-cell epitopes. The highspecificity and affinity of binding DARPins has been attributed rigidbody binding mode. Multi-specific or multi-valent constructs made bygenetic fusion show similar properties as single domain DARPins. Theabsence of cysteines in the scaffold enables engineering ofsite-specific cysteines, allowing site-directed coupling of chemicals tothe molecule.

In certain embodiments, the present invention provides a designedankyrin repeat protein (DARPin) comprising

(a) an N-Terminal Capping ankyrin repeat (AR),

(b) a C-Terminal Capping AR, and

(c) three to six AR modules of about 30 to 35 amino acids, wherein eachAR module binds with a target.

N-Terminal Capping AR (SEQ ID NO: 30) 5′-tTCCGCccatggACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTGTCTT CTAGgcggccgcCCCAAA-3′C-Terminal Capping AR (SEQ ID NO: 31) 5′-TTCCGCccatggTAGGAAGACCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAgcggccgcCCCAAA-3′

In certain embodiments, all of the AR modules are the same. In certainembodiments, one or more of the AR modules differ. In certainembodiments, an AR modules bind with TDP-43, tau, abeta oralpha-synuclein.

In certain embodiments, the DARPin is encoded by a sequence having atleast 90% sequence identity of any one of SEQ ID NO:7-28. In certainembodiments, the DARPin is encoded by a sequence having at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identityof any one of SEQ ID NO:7-28.

A “variant” of an amino acid sequence of a ligand or ligand fragmentdescribed herein, or a nucleic acid sequence encoding such an amino acidsequence, is a sequence that is substantially similar to SEQ ID NO:1-28.Variant amino acid and nucleic acid sequences include syntheticallyderived amino acid and nucleic acid sequences, or recombinantly derivedamino acid or nucleic acid sequences. Generally, amino acid or nucleicacid sequence variants of the invention will have at least 40, 50, 60,to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generallyat least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to SEQID NO: 1 -28.

The present invention includes variants of the amino acid sequences ofthe antibodies and antibody fragments described herein, as well asvariants of the nucleic acid sequences encoding such amino acidsequences (i.e., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID

NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, or SEQ ID NO:28).

“Variants” are intended to include sequences derived by deletion(so-called truncation) or addition of one or more amino acids to theN-terminal and/or C-terminal end, and/or addition of one or more basesto the 5′ or 3′ end of the nucleic acid sequence; deletion or additionof one or more amino acids/nucleic acids at one or more sites in thesequence; or substitution of one or more amino acids/nucleic acids atone or more sites in the sequence. The DARPins described herein may bealtered in various ways including amino acid substitutions, deletions,truncations, and insertions. Methods for such manipulations aregenerally known in the art. For example, amino acid sequence variantscan be prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. Thesubstitution may be a conserved substitution. A “conserved substitution”is a substitution of an amino acid with another amino acid having asimilar side chain. A conserved substitution would be a substitutionwith an amino acid that makes the smallest change possible in the chargeof the amino acid or size of the side chain of the amino acid(alternatively, in the size, charge or kind of chemical group within theside chain) such that the overall protein retains its spatialconformation but does not alter its biological activity. For example,common conserved changes might be Asp to Glu, Asn or Gln; His to Lys,Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanineis commonly used to substitute for other amino acids. The 20 essentialamino acids can be grouped as follows: alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan and methionine havingnonpolar side chains; glycine, serine, threonine, cystine, tyrosine,asparagine and glutamine having uncharged polar side chains; aspartateand glutamate having acidic side chains; and lysine, arginine, andhistidine having basic side chains.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

Nucleic Acids and Vectors

In certain embodiments, the present invention provides a nucleic acidencoding the DARPin described herein.

In certain embodiments, the present invention provides a vectorcomprising the nucleic acid described herein.

In certain embodiments, the vector is a pIT2 vector. In certainembodiments, the pIT2 vector lacks a BsaI restriction site. In certainembodiments, the vector lacks a PelB signal and comprises a DsbA signal.

In certain embodiments, the present invention provides a phagecomprising the vector described herein.

As used herein, the term “nucleic acid” and “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form, composed of monomers (nucleotides)containing a sugar, phosphate and a base that is either a purine orpyrimidine. Unless specifically limited, the term encompasses nucleicacids containing known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues.

A “nucleic acid fragment” is a portion of a given nucleic acid molecule.Deoxyribonucleic acid (DNA) in the majority of organisms is the geneticmaterial while ribonucleic acid (RNA) is involved in the transfer ofinformation contained within DNA into proteins. The term “nucleotidesequence” refers to a polymer of DNA or RNA which can be single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases capable of incorporation into DNA or RNA polymers.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidfragment,” “nucleic acid sequence or segment,” or “polynucleotide” mayalso be used interchangeably with gene, cDNA, DNA and RNA encoded by agene, e.g., genomic DNA, and even synthetic DNA sequences. The term alsoincludes sequences that include any of the known base analogs of DNA andRNA.

By “fragment” or “portion” is meant a full length or less than fulllength of the nucleotide sequence.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis that encode the native protein, as wellas those that encode a polypeptide having amino acid substitutions.Generally, nucleotide sequence variants of the invention will have in atleast one embodiment 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, atleast 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, to 98%, sequence identity to the native (endogenous) nucleotidesequence.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid sequences makes reference to a specified percentage ofresidues in the two sequences that are the same when aligned by sequencecomparison algorithms or by visual inspection.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences, wherein theportion of the polynucleotide sequence may comprise additions ordeletions (i.e., gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, or 79%; at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%; at least 90%, 91%, 92%, 93%, or 94%; or evenat least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to areference sequence using one of the alignment programs described usingstandard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched nucleic acid.Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl: T_(m) 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L. M is the molarity of monovalent cations, % GCis the percentage of guanosine and cytosine nucleotides in the DNA, %form is the percentage of formamide in the hybridization solution, and Lis the length of the hybrid in base pairs. T_(m) is reduced by about 1°C. for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T, those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T of less than 45° C. (aqueous solution) or 32°C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes. Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. and at least about 60° C. for longprobes (e.g., >50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide. Ingeneral, a signal to noise ratio of 2× (or higher) than that observedfor an unrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

“Operably-linked” nucleic acids refers to the association of nucleicacid sequences on single nucleic acid fragment so that the function ofone is affected by the other, e.g., an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. For example, a regulatory DNA sequence is said to be “operablylinked to” or “associated with” a DNA sequence that codes for an RNA ora polypeptide if the two sequences are situated such that the regulatoryDNA sequence affects expression of the coding DNA sequence (i.e., thatthe coding sequence or functional RNA is under the transcriptionalcontrol of the promoter). Coding sequences can be operably-linked toregulatory sequences in sense or antisense orientation. Control elementsoperably linked to a coding sequence are capable of effecting theexpression of the coding sequence. The control elements need not becontiguous with the coding sequence, so long as they function to directthe expression thereof. Thus, for example, intervening untranslated yettranscribed sequences can be present between a promoter and the codingsequence and the promoter can still be considered “operably linked” tothe coding sequence.

The terms “isolated and/or purified” refer to in vitro isolation of anucleic acid, e.g., a DNA or RNA molecule from its natural cellularenvironment, and from association with other components of the cell ortest solution (e.g. RNA pool), such as nucleic acid or polypeptide, sothat it can be sequenced, replicated, and/or expressed. For example,“isolated nucleic acid” may be a DNA molecule containing less than 31sequential nucleotides that is transcribed into an RNAi molecule. Suchan isolated RNAi molecule may, for example, form a hairpin structurewith a duplex 21 base pairs in length that is complementary orhybridizes to a sequence in a gene of interest, and remains stably boundunder stringent conditions (as defined by methods well known in the art,e.g., in Sambrook and Russell, 2001). Thus, the RNA or DNA is “isolated”in that it is free from at least one contaminating nucleic acid withwhich it is normally associated in the natural source of the RNA or DNAand is preferably substantially free of any other mammalian RNA or DNA.The phrase “free from at least one contaminating source nucleic acidwith which it is normally associated” includes the case where thenucleic acid is reintroduced into the source or natural cell but is in adifferent chromosomal location or is otherwise flanked by nucleic acidsequences not normally found in the source cell, e.g., in a vector orplasmid.

Nucleic acid molecules having base substitutions (i.e., variants) areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the nucleic acid molecule.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one of thesequences is affected by another. For example, a regulatory DNA sequenceis said to be “operably linked to” or “associated with” a DNA sequencethat codes for an RNA or a polypeptide if the two sequences are situatedsuch that the regulatory DNA sequence affects expression of the codingDNA sequence (i.e., that the coding sequence or functional RNA is underthe transcriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

As used herein, the term “derived” or “directed to” with respect to anucleotide molecule means that the molecule has complementary sequenceidentity to a particular molecule of interest.

In certain embodiments, the expression cassette further contains apromoter. In certain embodiments, the promoter is a regulatablepromoter. In certain embodiments, the promoter is a constitutivepromoter. In certain embodiments, the promoter is a PGK, CMV or RSVpromoter.

The present invention provides a vector containing the expressioncassette described above. Expression vectors include, but are notlimited to, viruses, plasmids, and other vehicles for deliveringheterologous genetic material to cells. Accordingly, the term“expression vector” as used herein refers to a vehicle for deliveringheterologous genetic material to a cell. In particular, the expressionvector is a recombinant adenoviral, adeno-associated virus, orlentivirus or retrovirus vector. In certain embodiments, the viralvector is an adenoviral, lentiviral, adeno-associated viral (AAV),poliovirus, HSV, or murine Maloney-based viral vector.

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. It also may include sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest. The expression cassette including thenucleotide sequence of interest may be chimeric. The expression cassettemay also be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. The expression ofthe nucleotide sequence in the expression cassette may be under thecontrol of a constitutive promoter or of a regulatable promoter thatinitiates transcription only when the host cell is exposed to someparticular stimulus. In the case of a multicellular organism, thepromoter can also be specific to a particular tissue or organ or stageof development.

Binding Molecules

As used herein, the term “binding molecule” includes antibodies, whichincludes scFvs (also called a “nanobodies”), humanized, fully human orchimeric antibodies, single-chain antibodies, diabodies, andantigen-binding fragments of antibodies (e.g., Fab fragments), andDARPins.

In certain embodiments, the binding molecule does not contain theconstant domain region of an antibody.

In certain embodiments, the binding molecule is less than 500 aminoacids in length, such as between 200-450 amino acids in length, or lessthan 400 amino acids in length.

In certain embodiments, the antibodies that are used in the presentinvention are those described in WO 2012/058308 (amyloid β-protein (Aβor beta amyloid)); WO 2012/082237 (amyloid β-protein (Aβ or betaamyloid)); WO 2014/059442 (tau).

In certain embodiments, the binding molecule specifically recognizesTDP-43 associated with frontotemporal dementia (FTD), but not TDP-43associated with amyotrophic lateral sclerosis (ALS) or TDP-43 associatedwith healthy human brain tissue.

In certain embodiments, the binding molecule that specificallyrecognizes TDP-43 associated with amyotrophic lateral sclerosis (ALS),but not TDP-43 associated with frontotemporal dementia (FTD) or TDP-43associated with healthy human brain tissue. In certain embodiments, thebinding molecule binds to TDP-43 associated with ALS and does not bindTDP-43 from healthy human brain tissue or TDP-43 associated with FTD. Incertain embodiments, the binding molecule comprises an amino acidsequence encoded by a nucleic acid, wherein the nucleic acid has atleast 80% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6.

SEQ ID NO: 1 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCCGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTTCTGCTTCTGGTACTTATACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTTCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATAATTATGCTCCTTATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ SEQ ID NO: 2 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTCGCCATGCTGGTCAGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATATGGCATCCCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCAGCGTACGAAGCCTCCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCC GC-3′SEQ ID NO: 3 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTGCTTCTGCTGGTACTGATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATACTACTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCAAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTACTTATGCTCCTGCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ SEQ ID NO: 4 5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTATTCTAGTCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ SEQ ID NO: 5 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAATAATGCTGGTATGATACAAATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATAATGCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATTCTGCTCCTGGTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ SEQ ID NO: 6 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATAATAGTGGTACTTCTACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTAATTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATGCTGCTGATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′

Detection Reagents and Assays

For purposes of the diagnostic methods of the invention, thecompositions or ligand of the invention (e.g., binding molecule such asan antibody or DARPin) may be conjugated to a detecting reagent thatfacilitates detection of the ligand. For example, example, the detectingreagent may be a direct label or an indirect label. The labels can bedirectly attached to or incorporated into the detection reagent bychemical or recombinant methods.

In one embodiment, a label is coupled to the ligand through a chemicallinker. Linker domains are typically polypeptide sequences, such as polygly sequences of between about 5 and 200 amino acids. In someembodiments, proline residues are incorporated into the linker toprevent the formation of significant secondary structural elements bythe linker. In certain embodiments, linkers are flexible amino acidsubsequences that are synthesized as part of a recombinant fusionprotein comprising the RNA recognition domain. In one embodiment, theflexible linker is an amino acid subsequence that includes a proline,such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about100. In other embodiments, a chemical linker is used to connectsynthetically or recombinantly produced recognition and labeling domainsubsequences. Such flexible linkers are known to persons of skill in theart. For example, poly(ethylene glycol) linkers are available fromShearwater Polymers, Inc. Huntsville, Ala. These linkers optionally haveamide linkages, sulfhydryl linkages, or heterofunctional linkages.

The detectable labels can be used in the assays of the present inventionto diagnose TBI, these labels are attached to the ligand of theinvention, can be primary labels (where the label comprises an elementthat is detected directly or that produces a directly detectableelement) or secondary labels (where the detected label binds to aprimary label, e.g., as is common in immunological labeling). Anintroduction to labels, labeling procedures and detection of labels isfound in Polak and Van Noorden (1997) Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. and in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals, a combinedhandbook and catalogue Published by Molecular Probes, Inc., Eugene,Oreg. Patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241.

Primary and secondary labels can include undetected elements as well asdetected elements. Useful primary and secondary labels in the presentinvention can include spectral labels such as green fluorescent protein,fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

Exemplary labels that can be used include those that use: 1)chemiluminescence (using horseradish peroxidase and/or alkalinephosphatase with substrates that produce photons as breakdown productsas described above) with kits being available, e.g., from MolecularProbes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL;2) color production (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored precipitate (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) fluorescence using, e.g., an enzyme such as alkaline phosphatase,together with the substrate AttoPhos (Amersham) or other substrates thatproduce fluorescent products, 4) fluorescence (e.g., using Cy-5(Amersham), fluorescein, and other fluorescent tags); 5) radioactivity.Other methods for labeling and detection will be readily apparent to oneskilled in the art.

Where the ligand-based compositions of the invention are contemplated tobe used in a clinical setting, the labels are preferably non-radioactiveand readily detected without the necessity of sophisticatedinstrumentation. In certain embodiments, detection of the labels willyield a visible signal that is immediately discernable upon visualinspection. One example of detectable secondary labeling strategies usesan antibody or DARPin that recognizes oligomers in which the antibody orDARPin is linked to an enzyme (typically by recombinant or covalentchemical bonding). The antibody or DARPin is detected when the enzymereacts with its substrate, producing a detectable product. In certainembodiments, enzymes that can be conjugated to detection reagents of theinvention include, e.g., β-galactosidase, luciferase, horse radishperoxidase, and alkaline phosphatase. The chemiluminescent substrate forluciferase is luciferin. One embodiment of a fluorescent substrate forβ-galactosidase is 4-methylumbelliferyl-β-D-galactoside. Embodiments ofalkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP),which is detected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer,and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′ diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

The presence of a label can be detected by inspection, or a detectorwhich monitors a particular probe or probe combination is used to detectthe detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

The ligand compositions of the invention can be used in any diagnosticassay format to determine the presence of tau oligomers. A variety ofimmunodetection methods are contemplated for this embodiment. Suchimmunodetection methods include enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), immunoradiometric assay,fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, andWestern blot, though several others are well known to those of ordinaryskill. The steps of various useful immunodetection methods have beendescribed in the scientific literature.

In general, the binding methods include obtaining a sample suspected ofcontaining a protein, polypeptide and/or peptide (e.g., TDP-43), andcontacting the sample with a first antibody, monoclonal or polyclonal,or DARPin, in accordance with the present invention, as the case may be,under conditions effective to allow the formation of complexes.

The binding methods include methods for detecting and quantifying theamount of the target oligomer component in a sample and the detectionand quantification of any complexes formed during the binding process.Here, one would obtain a sample suspected of containing targetoligomers, and contact the sample with an antibody fragment or DARPin ofthe invention, and then detect and quantify the amount of complexesformed under the specific conditions.

Contacting the chosen biological sample with the antibody or DARPinunder effective conditions and for a period of time sufficient to allowthe formation of complexes (primary complexes) is generally a matter ofsimply adding the antibody composition to the sample and incubating themixture for a period of time long enough for the antibodies or DARPin toform complexes with, i.e., to bind to, any antigens present. After thistime, the sample-antibody composition, such as a tissue section, ELISAplate, dot blot or western blot, will generally be washed to remove anynon-specifically bound antibody species, allowing only those scFvmolecules specifically bound within the primary complexes to bedetected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

As noted above, a ligand of the invention may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary complexes in the compositionto be determined. Alternatively, a first antibody that becomes boundwithin the primary complexes may be detected by means of a secondbinding ligand that has binding affinity for the complex. In thesecases, the second binding ligand may be linked to a detectable label.The second binding ligand is itself often an antibody, which may thus betermed a “secondary” ligand. The primary complexes are contacted withthe labeled, secondary binding ligand or antibody under effectiveconditions and for a period of time sufficient to allow the formation ofsecondary complexes. The secondary complexes are then generally washedto remove any non-specifically bound labeled secondary antibodies orligands, and the remaining label in the secondary complexes is thendetected.

Further methods include the detection of primary complexes by a two-stepapproach. A second binding ligand, such as an antibody, that has bindingaffinity for the scFV (e.g., F9T or D11C) is used to form secondarycomplexes, as described above. After washing, the secondary complexesare contacted with a third binding ligand or antibody that has bindingaffinity for the second antibody, again under effective conditions andfor a period of time sufficient to allow the formation of complexes(tertiary complexes). The third ligand or antibody is linked to adetectable label, allowing detection of the tertiary complexes thusformed. This system may provide for signal amplification if this isdesired.

One method of immunodetection designed by Charles Cantor uses twodifferent antibodies. A first step biotinylated, monoclonal orpolyclonal antibody (in the present example a scFv or DARPin) is used todetect the target antigen(s), and a second step antibody is then used todetect the biotin attached to the complex. In this method the sample tobe tested is first incubated in a solution containing the first stepligand. If the target antigen is present, some of the ligand binds tothe antigen to form a biotinylated ligand/antigen complex. Theligand/antigen complex is then amplified by incubation in successivesolutions of streptavidin (or avidin), biotinylated DNA, and/orcomplementary biotinylated DNA, with each step adding additional biotinsites to the ligand /antigen complex. The amplification steps arerepeated until a suitable level of amplification is achieved, at whichpoint the sample is incubated in a solution containing the second stepantibody against biotin. This second step antibody is labeled, as forexample with an enzyme that can be used to detect the presence of theantibody/antigen complex by histoenzymology using a chromogen substrate.With suitable amplification, a conjugate can be produced which ismacroscopically visible.

Another known method of detection takes advantage of the immuno-PCR(Polymerase Chain Reaction) methodology. The PCR method is similar tothe Cantor method up to the incubation with biotinylated DNA, however,instead of using multiple rounds of streptavidin and biotinylated DNAincubation, the DNA/biotin/streptavidin/antibody complex is washed outwith a low pH or high salt buffer that releases the antibody. Theresulting wash solution is then used to carry out a PCR reaction withsuitable primers with appropriate controls. At least in theory, theenormous amplification capability and specificity of PCR can be utilizedto detect a single antigen molecule.

As detailed above, the assays in their most simple and/or direct senseare binding assays. Certain preferred assays are the various types ofenzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays(RIA) known in the art. Immunohistochemical detection using tissuesections is also particularly useful. However, it will be readilyappreciated that detection is not limited to such techniques, and/orwestern blotting, dot blotting, FACS analyses, and/or the like may alsobe used.

The diagnostic assay format that may be used in the present inventioncould take any conventional format such as ELISA or other platforms suchas luminex or biosensors. The present invention provides various ligands(e.g., DARPins). These ligands can readily be modified to facilitatediagnostic assays, for example a tag (such as GFP) can be added to theseligands to increase sensitivity. In one exemplary ELISA, ligand (e.g.,antibodies or DARPins) are immobilized onto a selected surfaceexhibiting protein affinity, such as a well in a polystyrene microtiterplate. Then, a test composition suspected of containing a targetoligomer, such as a clinical sample (e.g., a biological sample obtainedfrom the subject), is added to the wells. After binding and/or washingto remove non-specifically bound complexes, the bound antigen may bedetected. Detection is generally achieved by the addition of an antibodythat is linked to a detectable label. This type of ELISA is a simple“sandwich ELISA.” Detection may also be achieved by the addition of asecond antibody, followed by the addition of a third antibody that hasbinding affinity for the second antibody, with the third antibody beinglinked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withbinding agents. After binding and/or washing to remove non-specificallybound complexes, the bound anti-binding agents are detected. Where theinitial binding agents are linked to a detectable label, the complexesmay be detected directly. Again, the complexes may be detected using asecond antibody that has binding affinity for the first binding agents,with the second antibody being linked to a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodies(or nanobodies or DARPins) against an antigen are added to the wells,allowed to bind, and/or detected by means of their label. The amount ofan antigen in an unknown sample is then determined by mixing the samplewith the labeled antibodies against the antigen during incubation withcoated wells. The presence of an antigen in the sample acts to reducethe amount of antibody against the antigen available for binding to thewell and thus reduces the ultimate signal. This is also appropriate fordetecting antibodies against an antigen in an unknown sample, where theunlabeled antibodies bind to the antigen-coated wells and also reducesthe amount of antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.

In coating a plate with either target oligomers or a ligand (e.g.,antibody or DARPin) of the invention, one will generally incubate thewells of the plate with a solution of the antigen or ligand, eitherovernight or for a specified period of hours. The wells of the platewill then be washed to remove incompletely adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein orsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand. “Under conditions effective to allowimmune complex (antigen/antibody) formation” means that the conditionspreferably include diluting the tau oligomers and/or scFv compositionwith solutions such as BSA, bovine gamma globulin (BGG) or phosphatebuffered saline (PBS)/Tween. These added agents also tend to assist inthe reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An example of a washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. This may be an enzyme that willgenerate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

Diagnostic Methods

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject by measuring levels of TDP-43, tau, abeta and/oralpha-synuclein at two or more time points. In certain embodiments, one,two, three or all four proteins will be measured to determine differenttypes of damage that has occurred. It would be expected that increaseddamage and increased risk of further neurodegenerative disorders areindicated by higher concentrations of the different toxic proteinvariants for longer periods of time after injury. Different proteinvariants will indicate different types of damage and likely indicateincreased risk for different neurodegenerative disorders. Some patientsmay have only one protein, others multiple or all four. Certaincombinations are likely to lead to certain disorders (for example, abetaand tau would indicate Alzheimer's, and a-syn and tau would indicateParkinson's), but they all indicate brain injury. Protein concentrationtrajectories are key, as higher concentrations of the proteins forlonger times is detrimental; levels that go down quickly are good; andlevels that increase would be very bad. It can be beneficial, thereforeto measure levels over a period of time for further predictive accuracy.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at two or more timespost-injury;

(B) assessing protein levels of toxic variants of TDP-43, tau, abetaand/or alpha-synuclein in the sample by detecting protein levels oftoxic variants of TDP-43, tau, abeta and/or alpha-synuclein in thesamples;

(C) comparing the protein levels of toxic variants of TDP-43, tau, abetaand/or alpha-synuclein protein levels in the sample at each time pointwith protein levels of toxic variants of TDP-43, tau, abeta and/oralpha-synuclein in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having elevated protein levels of toxic variants ofTDP-43 tau, abeta and/or alpha-synuclein has a high risk of TBI.

In certain embodiments, a sample is obtained from the subject within 6hours post-injury.

In certain embodiments, a sample is obtained from the subject about 12to 36 hours post-injury.

In certain embodiments, a sample is obtained from the subject about 5 to10 days post injury.

In certain embodiments, a sample is obtained from the subject about 2 to4 weeks days post injury.

In certain embodiments, the sample and the normal control are bloodproduct samples or cerebrospinal fluid (CSF) samples. In certainembodiments, the blood product is serum.

In certain embodiments, the detecting in step (B) is by means of aligand specific for the protein.

In certain embodiments, the ligand is an antibody.

In certain embodiments, the ligand is a designed ankyrin repeat protein(DARPin).

In certain embodiments, the protein levels are detected by means ofELISA.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing protein levels of a toxic variant of TDP-43 in the sampleby detecting protein levels of toxic variants of TDP-43 in the samples;

(C) comparing the protein levels of toxic variants of TDP-43 in thesample at each time point with protein levels of toxic variants ofTDP-43 in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having increased protein levels of toxic variants ofTDP-43 in the samples at all four time points than those of the normalcontrol, and having a decreased level of the toxic variants of TDP-43 atthe 24-hour time point as compared to the 6-hour time point, having adecreased level of TDP-43 at the 5-day time point as compared to the24-hour time point, and having an increased level of TDP-43 at the10-day time point as compared to either the 24-hour or 5-day time pointhas a high risk of TBI.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing protein levels of a toxic variant of tau in the sample bydetecting the protein levels of the toxic variant of tau protein levelsin the samples;

(C) comparing the protein level of the toxic variant of tau in thesample at each time point with the protein level the toxic variant oftau in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having an increased protein level of the toxic variantof tau in the sample at the 24-hour time point and at the 5-day timepoint as compared to that of the normal control, having comparableprotein levels of the toxic variant of tau at the 6-hour and 10-day timepoints as compared to that of the normal control, and having anincreased protein level of the toxic variant of tau at the 5-day timepoint as compared to the 24-hour time point has a high risk of TBI.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing protein levels of a toxic variant of abeta in the sampleby detecting protein levels of the toxic variant of abeta in thesamples;

(C) comparing protein levels of the toxic variant of the abeta in thesample at each time point with abeta protein levels in a normal control;and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having an increased protein levels of the toxicvariant of abeta in the sample at the 24-hour time point, the 5-day timepoint and the 10-day time point as compared to that of the normalcontrol, having comparable protein level of the toxic variant of abetaat the 6-hour as compared to that of the normal control, and having anincreased protein level of the toxic variant of abeta at the 5-day timepoint as compared to the 24-hour time point and 10-day time point has ahigh risk of TBI.

In certain embodiments, the present invention provides a method fordetermining risk of traumatic brain injury (TBI), assessment of theamount of neuronal damage, and/or susceptibility to neurodegenerativedisease in a subject, comprising the steps of:

(A) providing samples obtained from a subject at about 6-hours, about24-hours, about 5-days and about 10-days post-injury;

(B) assessing protein levels of a toxic variant of alpha-synuclein inthe sample by detecting protein levels of the toxic variant ofalpha-synuclein in the samples;

(C) comparing the protein levels of the toxic variant of alpha-synucleinin the sample at each time point with protein levels of the toxicvariant of alpha-synuclein in a normal control; and

(D) determining whether the subject has a risk of TBI in accordance withthe result of step (C);

wherein a subject having an increased protein levels of the toxicvariant of alpha-synuclein in the sample at the 5 day time point, the10-day time point as compared to that of the normal control, havingcomparable protein level of the toxic variant of alpha-synuclein at the6-hour time point and 24-hour time point as compared to that of thenormal control, and having an increased protein level of the toxicvariant of alpha-synuclein at the 5-day time point as compared to the10-day time point has a high risk of TBI.

In certain embodiments, the sample and the normal control are bloodproduct samples or cerebrospinal fluid (CSF) samples. In certainembodiments, the blood product is serum.

In certain embodiments, the detecting in step (B) is by means of aligand specific for the protein. In certain embodiments, the ligand isan antibody. In certain embodiments, the ligand is a designed ankyrinrepeat protein (DARPin).

In certain embodiments, the protein levels are detected by means ofELISA.

EXAMPLE 1 DARPins and Traumatic Brain Injury

The morphology specific reagents described above were selected fromantibody fragment (scFv) libraries. ScFvs are susceptible to misfoldingand aggregation which makes them difficult to produce in large scale.Designed ankyrin repeat proteins (DARPins) have been suggested as morestable alterative to scFvs. To isolate DARPins against several proteinvariants, a new DARPin library as generated. In the present library, theproteins contained an N-Terminal capping AR, one AR module and theC-Terminal capping AR. In the AR module, there were seven locationswhere the amino acids were varied.

This DARPin library was utilized in various AFM based biopannings toisolate antibodies against several protein variants. One set ofbiopanning was conducted against any protein variant present in bothbrain tissue and sera from individuals with Alzheimer's disease (AD).ELISA analysis of one of these clones with the TBI cases revealedreactivity at 24 hours, 5 days and 10 days, with reactivity decreasingafter 5 days (FIG. 1). We are in the initial stages of characterizingthe clones from this and the other DARPin biopannings, but results thusfar indicate a preference for disease tissue over healthy controls.Since these panning were not against a specific known target, we stillneed to identify the protein target by mass spectrometry. However,clearly the DARPin library is a valuable resource to isolate DARPinsthat selectively bind protein variants associated with TBI. The DARPinshown here has a different reactivity profile than the protein variantsdiscussed above as the DARPin shows high reactivity at the early timepoints and then decreases.

EXAMPLE 2 Generation of a DARPin Library of 10¹⁰ Diversity Consisting ofa Core of 3-4 Repeat Ankyrin Units with a Randomized 7 Amino AcidBinding Loop

A designed ankyrin repeat protein (DARPin) N1C library was generated byligating the N1 product with a SRP modified pIT2 vector containing theC-Terminal capping AR. After the ligation procedure, throughelectroporation the N1C library was produced. Titration of the libraryindicated that there were 10¹⁰ cells/ml. DNA sequencing of 20 clonesrevealed 45% of the clones were correct and each had a different ARmodule (SEQ ID NOs: 7-15), 25% had errors in the N-Cap or pre-N-Cap, 20%had errors in the N-Cap, and AR module (SEQ ID NOs: 16-20), and 10% wereempty. A library consisting of 3-4 AR module repeats was the desiredsize. After multiple ligation attempts and some difficulty in ligatingthese AR repeats together, phage were produced from the N1C library andthese phage particles were used in the panning procedures. Phageparticles were produced in a manner similar to the steps outlined in theTomlinson I and J protocols (world-wide-web atlifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf).

N1C Library Generic Sequence (SEQ ID NO: 29) 5′-gctagcATGAAAAAGATTTggctggcgctggctggTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAggcccagccggccATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTNNNGACNNNNNNGGTNNNACTCCGCTGCACCTGGCTGCTNNNNNNGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGHACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAgcggccgcACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGG-3′The 9 of 20 DARPins that were correct when sequenced are below (45%).DARPin 1 (SEQ ID NO: 7) 5′-GATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGACGACTACCGTGGTTCTACTCCGCTGCACCTGGCTGCTATGGCTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGCACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAANAATAGGTTCCGAANTAGGCAGGGNGCATTNACTGTTTATACGGGCACTGTTACTCNNGGCACTGACCCCGTTAAAACTTATTANCAGTANNNTCCTGTATCATCAAAAGNCATGTATGANGCNTNCTGGNNCNGNAANTCNNANACTG-3′ DARPin 2 (SEQ ID NO: 8) 5′-CTATTTCAGGAGANAGTCATAGCTAGCNTNNNNNNNANTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGTTGACCGTAAAGGTAACACTCCGCTGCACCTGGCTGCTCAGTACGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGCACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAANAATAGGTTCCGAAATANNCAGGGTGCATTAACTGTTTATACGGGCACTGNTACTCANGGCACTGACCCCGTNAAAACTT-3′ DARPin 3 (SEQ ID NO: 9) 5′-GATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGTTGACGCNCANGGTACTACTCCGCTGCACCTGGCTGCTCGNNNNNGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATNNNCAGGGNGCATTNACTGTTTATACGGGCACTGNTACTCNNGGNNCTGACCCCGNTAAAACTTATTANCAGTANNCTCCTGTATCATCAAAAGCCATGTATGACGCTT-3′ DARPin 4(SEQ ID NO 10) 5′-TTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTANNGACCANNNNGGTNNTACTCCGCTGCACCTGGCTGCTNGGNNTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGANCTGGCTGAAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGANGATCTGAATGGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGANGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGANGGTGGCGGTTCTGANGGTGGCGGTTCTGANGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACNGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTNGA GGAGTCTCAG-3′DARPin 5 (SEQ ID NO: 11) 5′-TTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTAACGACATCGAAGGTCATACTCCGCTGCACCTGGCTGCTATCTACGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCANAANAATAGGTTCCGAAATNGNCAGGGTGCATTNACTGTTTATACGGGCACTGNTACTCNNGGCACTGACCCNGTNAAAACTTATTACCAGTACNCTCCTGTATCATCAAAAGCCAT-3′ DARPin 6 (SEQ ID NO: 12) 5′-TTCTATTTCAGGAGANAGTCATAGCTAGCATGAAAAAGANTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTAAAGACCATGAAGGTCAGACTCCGCTGCACCTGGCTGCTCAGATCGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCANAANAATAGGTTCCGAAATAGGCAGGGTGCATTNACTGTTTATACGGGCACTGTNACTCANGGCACTGACCCCGTTAAAACTTATTACCAGTACNCTCCTGTATCATCAAAAGNCATGTATGA-3′ DARPin 7 (SEQ ID NO: 13) 5′-TATTTNAGGAGANAGTCATAGCTAGCATGNAAAAGANTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTATGGACAACGCTGGTACTACTCCGCTGCACCTGGCTGCTCAGTTCGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATANNTTCCGAAATAGGCAGGGTGCATTAACTGTTTANACGGGCACTGNNACTCANGGNACTGACCCCGNNAAAACTTATTACCAGTACACTNCTGTATCATCAAANCCATGNATGA-3′ DARPin 8 (SEQ ID NO: 14) 5′-TTCTATTTCAGGAGANAGTCATAGCTAGCATGAAAAAGANTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTNGGGACCTGNNNGGTACNACTCCGCTGCACCTGGCTGCTANNGNNGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCANAATAATANNTTCCNAAATNGNCAGGGTGCATTNACTGTTTATACNGGCNCTGNNACTCNNGGNACTGACCCCGTTAAAACT-3′ DARPin 9 (SEQ ID NO: 15) 5′-GAGANAGTCATAGCTAGCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCCGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTAACGACCTGGAAGGTGACACTCCGCTGCACCTGGCTGCTTACATCGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGANGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCANAANAATNNNTTCCGAAATNNNCAGGGTGCATTAACTGTTTATACGGGCACTGNTACTCAAGGCACTGACCCCGT-3′The 5 of 20 DARPins that had errors in the N-Cap orpre-N-Cap are below (25%). DARPin 10 (SEQ ID NO: 16) 5′-ATTTGGCTGGCGCTGGCTGGTTNAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGACGTTAACGCTATGGACGCTTACGGTAACACTCCGCTGCACCTGGCTGCTTGGTCTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTNGNNCNGTAAANTCANAGACTGCGCTTTNCATTCTGGCTTTNATGANGNTNCATTCNTTTGTGAATA T-3′ DARPin 11(SEQ ID NO: 17) 5′-TTCGCCACNTNTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTANNNAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCNTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGACGTTAACGCTTACGACCGTGTTGGTGAAACTCCGCTGCACCTGGCTGCTGACAACGGTCACCCGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATC-3′ DARPin 12 (SEQ ID NO: 18)5′- GGGGAAACNNCTGGTATNTTTATAGNCCTGTCGGGTTTCGCCACCTNTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGNCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTCCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCAAATTCTATTTCAAGGAGACAGTCATAGCTAGCATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGACGTTAACGCTCGTGACATGACTGGTTGGACTCCGCTGCACCTGGCTGCTACTACTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCNTCTCAGAAGAGGATC-3′ DARPin 13 (SEQ ID NO: 19) 5′-CTATTTCNGGAGANAGTCATAGCTAGCATGNAAAAGANTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGACGTTAACGCTTGGGACGTTCATGGTGACACTCCGCTGCACCTGGCTGCTATGGACGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATNNNCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTNCTGTATCATCAAAAGCCNTGTATGACGCTNACTGGNNCNGNAAANTCNNANACTGCNCTTTNCAT-3′ DARPin 14 (SEQ ID NO: 20) 5′-TTCTATTTCNGGAGANNGTCATAGCTAGCATGAAAAAGANTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGGCGGAACAGCAGTTTCTTACCCAGGTCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGAAGACTACCAGGGTCTGACTCCGCTGCACCTGGCTGCTGACACTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGCACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCNTAATACTTTCATGTTTCA-3′ ALS DARPin Clone(SEQ ID NO: 21) 5′-TTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGTTGACATGTACGGTATCACTCCGCTGCACCTGGCTGCTGAATACGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGNACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTNATACTTTCATGTTTCANAA-3′ PD DARPin Clone (SEQ ID NO: 22) 5′-AAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGCTGACGTTAAAGGTGAAACTCCGCTGCACCTGGCTGCTTGGGACGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGNTTCANAATAATAGGTTCCGAAATAGGCAGGGNGCATTNNNTGNTTANACGGGNNCNNNTACTCNNGGCACTGACCCCGTNAANC- 3′AD DARPin Clone 1 (SEQ ID NO: 23) 5′-AAGATTTGGCTGGCGCTGgctggNTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGACGACCGTAACGGTATGACTCCGCTGCACCTGGCTGCTCATCAGGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCNNAATAATAGGTTCCGAAATAGGCAGGGTGCATNNANTGTTTANACGGNCACTGNTACTCNAGGCACTGACCCCGTTAAACTTATTACCAGNANNCTCCTGTATCATCAAAAGCCATGTATGACGCT-3′ AD DARPin Clone 2(SEQ ID NO: 24) 5′-TGGCTGGCGCTGgctggTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTAAAGACTCTGTTGGTAAAACTCCGCTGCACCTGGCTGCTCATTGGGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGCACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCNNNNACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGANGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACNGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGNNNGTCTCAGCCTCTTATACTTTCATGTTTCANAATAATAGGTNNCGAAANAGGCAGGGTGCATTAACTGTTTATANNGGCACTGNTANNCANGGNACTGACCCCNTNAAACTTATTACCAGT- 3′AD DARPin Clone 3 (SEQ ID NO: 25) 5′-TGGNTGGCGCTGgtggNTTAgTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCTGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTACTGACACTGACGGTTCTAGTCCGCTGCACCTGGCTGCTCAGGAAGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCANTNNCTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGANGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCANAATAATNNNTTCCGAAATAGGCAGGGTGCATTAACTGTTTATACGGGCACTGTTACTCANGGCACTGACCCCGTTAAACTTATTACCA G-3′AD DARPin Clone 4 (SEQ ID NO: 26) 5′-GGCTGGCGCTGgctggttTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTGCTGACTTCAACGGTCAAACTCCGCTGCACCTGGCTGCTGTTTGGGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGAACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCNNNNACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGANGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCANNCTCTTAATACTTTCATGTTTCAGAATAATANNNTCCGAAATAGG-3′ AD DARPin Clone 5(SEQ ID NO: 27) 5′-TTTGGCTGGCGCTGNNNNTTTAGTTTTAGCGTTTAGCGCATCGGCGGACTACAAAGAGGCCCAGCCGGCCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTCGTGACGTTTCTGGTGCTACTCCACTGCACCTGGCTGCTACTTGGGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTNANNNACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCNTGTTTCANAATAATAGGTTCCGAAATAGGNAGGGTGCATTAACTGTTTATACGGGCACTNNTACTCANGCANTGNCCCCGTNAAACTTNTACC AGT-3′AD DARPin Clone 6 (SEQ ID NO: 28) 5′-TTTAGCGCATCGGCGGACTACAAAGNNNNNNNNNNGGNCATGGACCTGGGTAAGAAACTGCTGGAAGCTGCTCGTGCTGGTCAGGACGACGAAGTTCGTATCCTGATGGCTAACGGTGCTGACGTTAACGCTCGTGACGTTACTGGTGTTACTCCGTTGCACCTGGCTGCTAACCGTGGTCACCTGGAAATCGTTGAAGTTCTGCTGAAGTACGGTGCTGACGTTAACGCTCAGGACAAATTCGGTAAGACCGCTTTCGACATCTCCATCGACAACGGTAACGAGGACCTGGCTGAAATCCTGCAAGCGGCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCNGNNTGGGGCCGCATAGACTGTTGAAAGTTGTTTAGCAAAACCTCATACAGAAAATTCNANNACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTGGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGNTCTGAGGGTGGCGGTTCTGAGGNTGGCGGNTCTGANGGTGGCGGTACTAAANCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGANNGCACTTATCCGCCTNNNACTGAGCAAAACCCCGCTAATCCTAATCCTTCNCTTGAGGAGNCTCAGCCTCTTAATACTTTNNTGNTNCAGANNATAGGTTCCGAAANTNGGNAGGNNGCATTAACTGTTTNNACGGGCNCTGNTNCTTCAAGGCACTGANCCCGTTA-3′

The following is a description of the plasmid and library construction.When generating the DARPin library the pIT2 phagemid is used. BsaI isone of the restriction enzymes used to assemble the DARPin library andit was discovered that the restriction site for this enzyme was alsopresent on the pIT2 vector. To remedy this problem, a PCR product wasfirst produced using primers that mutated one of the bases in the BsaIrestriction site. This PCR product was then used in a second reactionalong with another primer to generate the new pIT2 vector lacking theBsaI restriction site.

It was observed that the expression of the DARPin library improved whenusing the signal recognition particle (SRP) translocation pathway. ThepIT2 vector has the PelB signal sequence and so the PelB was changed tothe DsbA signal sequence. The DsbA signal was generated using assemblyPCR with four oligonucleotides. To facilitate the replacement of PelBsignal with the DsbA signal, an additional restriction site was alsoadded to the pIT2 vector lacking the BsaI site. After transformation ofthis SRP modified BsaI lacking pIT2 vector into TG1 competent cells,some of the clones were sequenced and the results indicated that PelBwas replaced by the DsbA signal.

To ensure that the DsbA signal allows for correct expression ofproteins, this modified pIT2 vector was digested with NcoI and NotIrestriction enzymes and inserted two different scFvs that werepreviously isolated. The c-myc tag on the scFv was used in the Westernblotting process to determine if the scFv of interest was produced. Theresults indicated that the SRP modified pIT2 vector was able to produceboth scFvs. One of the scFvs was purified using Nickel bead purificationto ensure the scFv could be purified. Western blotting showed bands atthe correct location for the scFvs. The two scFvs were inserted into thevector in order to have better protein production results with one ofthe scFvs as compared to the other. The production of both scFvs wassatisfactory. Since the SRP modified vector was able to produceproteins, protein expression is attainable in the DARPin library.

Next, it was verified that the SRP modified pIT2 vector displayed theproteins on the phages, since the DARPin library is used in thebiopanning process. So again, using the NcoI and NotI restriction sites,another scFv was inserted into the vector. Using the Tomlinson I and Jlibrary protocol, phage displaying this scFv was produced. The titer ofthe phage produced was good. These phages were then used in an ELISA todetermine binding to it corresponding targets. Based on the bindingresults of the ELISA, the scFv was being displayed properly. Therefore,the new SRP modified pIT2 vector was also able to display proteinscorrectly in the phage display process.

To produce the AR proteins, the N-Terminal capping AR, the AR modulesand the C-Terminal capping AR were need. The N-Terminal capping AR andC-Terminal capping AR were generated using assembly PCR. The C-Terminalcapping AR was then cloned into the SRP modified pIT2 vector since oncethe N-Terminal capping AR and the AR modules are ligated together, theyare cloned into this vector containing the C-Terminal capping AR. TheN-Terminal capping AR was also cloned into the SRP modified pIT2 vectorto verify the correct sequence was assembled in the PCR. This vector wasthen used with the N-Terminal capping AR and two primers to producelarge number of copies of this sequence rather than using the productsgenerated from assembly PCR to ligate the N-Terminal capping AR to theAR modules, so as to reduce any errors in the N-Terminal capping ARregion that may occur during assembly PCR.

Using six oligonucleotides the AR module was assembled, and based on DNAgel electrophoresis, the product produced was the correct size. The bandwas then gel extracted. The AR module and the N-Terminal capping AR weredigested and ligated together and the product (labeled as the N1) wasrun on a DNA gel. The band produced was present at the correct locationon the gel. Large quantities of the SRP modified pIT2 vector containingthe C-Terminal capping AR were produced. This vector and the N1 productwere digested with the appropriate restriction enzymes and these twoproducts are ligated together.

These vectors are transformed into TG1 cells to produce the complete N1Clibrary. Once the N1C library is generated, the sequences are checked toensure correct assembly of the N-Terminal capping AR, the AR module, andthe C-Terminal capping AR. The N1 product is amplified and more ARmodules are added to N1 to generate the N2, N3 and N4 products, whichare ligated into the vector containing the C-Terminal capping AR toproduce the N2C, N3C and N4C libraries, respectively. These librariesare then used in the biopanning process to isolate DARPin proteins thatspecifically recognize different morphologies of a-synuclein using thenovel AFM/phage display-based biopanning techniques.

EXAMPLE 3 Isolation of DARPin Proteins that Specifically RecognizeDifferent Morphologies of A-Synuclein Using Novel AFM/Phage DisplayBased Biopanning Techniques

The role of TDP-43 in Amyotrophic Lateral Sclerosis (ALS) has been ofinterest. Reagents are developed that are reactive with the variousforms of TDP-43 present in the brain of individuals with ALS, as well asthe detection of such targets in cerebrospinal fluid (CSF) and serasamples. A N1C phage library was used to isolate such reagents againstTDP-43, and the library was compared with a commercially available scFvlibrary. A schematic summarizing the details of the DARPin ALSbiopanning procedures is illustrated in FIG. 2.

Atomic force microscopy (AFM) was used in the biopanning protocol toprovide two main advantages, including the verification of successfulremoval of off-targets and secondly to reduce the required quantity ofthe target antigen (whether purified or in a crude mixture). Briefly, 23immunotubes were coated with 1 mg/ml of bovine serum albumin (BSA). TheBSA solution from tube #1 was removed, the tube washed and the DARPinlibrary added. The tube was allowed to rotate for 30 minutes. The BSAwas removed from the second tube and the phage transferred to the secondtube from the first. This was repeated for all 23 tubes. In the past 12immunotubes were sufficient to removed negative binders to BSA. However,using AFM visualization showed that phages binding to BSA were stillvisible after 12 rounds. Only after 23 rounds of negative panning (NP)were the reagents reactive to BSA removed. Utilizing the phage after the23 rounds of NP against BSA, reactivity to healthy human tissue (HT) wasvisible. The healthy human tissue is a mixture of five samples from themotor cortex, two samples from the superior frontal gyrus (SFG) andthree from the middle temporal gyrus (MTG). After 12 rounds of NPagainst HT using immunotubes, the phages that were reactive to HT wereremoved. TDP-43 also plays a role in some cases of FrontotemporalDementia (FTD) and any potential ALS clones that are cross-reactive toFTD are less beneficial as an ALS diagnostic or therapeutic reagent.Thus using a combination of six FTD human tissue samples from the motorcortex, reactivity of the phages after the NP against HT to FTD tissuewere examined. Phage binding was visible, but not after eight rounds ofNP against FTD tissue using immunotubes. TDP-43 may also play a role inother neurodegenerative diseases such as Alzheimer's (AD) andParkinson's (PD).

Using seven AD human brain tissue samples from the MTG and seven fromthe SFG and the phage after the NP against FTD tissue, reactivity wasvisible. After eight rounds of NP against the AD tissue usingimmunotubes no reactivity was visible. For the NP against PD human braintissue nine samples from the MTG were utilized with varying p129-synlevels. After eight rounds of NP against AD tissue the phage displayedreactivity to PD tissue. After eight rounds of NP against PD tissueusing immunotubes reactivity was not detected. Since the protein TDP-43is of particular interest, phages were removed that would bind healthyTDP-43 variants as well. TDP-43 was immunoprecipitated from the brain ofa healthy individual and employed in three rounds of negative panningusing mica (instead of immunotubes due to limited sample quantity). Micawas utilized for the remaining panning steps. TDP-43 was alsoimmunoprecipitated from the brains of three individuals with ALS andthree with FTD. After the NP against healthy TDP-43, two rounds of NPagainst FTD TDP-43 were carried out. Some of the phages were then usedin one round of positive panning (PP) against ALS TDP-43 and forcomparison one round of PP against ALS human brain tissue (a combinationof 5 samples from the motor cortex) was also performed. Numerous cloneswere eluted from both positive Panning steps. To reduce the quantity ofclones needed for assessments in the further characterization studies,some of the remaining phage was used after the two rounds of NP againstthe FTE TEP-43 and one more round of PP against ALS TDP-43 wascompleted. The phages that bound were eluted with glycine allowing theantibody to remain attached to the phage (normally, trypsin elution isemployed but was not here in order to avoid cleavage of the phagearticle). The eluted phages were then added to mica with the ALS tissuemixture and the bound phages eluted. From this procedure two clones wereeluted. The intent in performing this 2-step PP procedure was that theclones obtained are reactive with both the TDP-43 protein and the ALStissue. Tissue reactivity was ensured in the event that some forms tothe immunoprecipitated TDP-43 proteins were altered during theimunoprecipitation process. DNA sequencing of the two clones revealedone clone with no errors (complete protein sequence). This clone ischaracterized in future studies for ALS specificity. Clones were alsoobtained that bound only to the ALS TDP-43 or ALS Tissue. (FIG. 2)

Reviewing the schematic in FIG. 2, it is noted that extensive negativebiopanning procedures were carried out to obtain the potential ALSTDP-43 DARPin clones. After each set of NP against a particular target asmall quantity of phage was saved in case of contamination. Some ofthese phages were used in other PP procedures to isolate potentialDARPin clones against other targets of interest. First, potentialDARPins against protein variants present in both PD human brain tissueand sera samples were examined.

FIG. 3 highlights the procedures completed to obtain the potential PDspecific DARPins. The phages were selected that remained after eightrounds of NP against AD human brain tissue (since this is before phageswere removed with PD tissue). All these panning steps were achievedusing mica. First, two rounds of NP were carried out against health sera(mixture of five samples) and two rounds of NP against AD sera (mixtureof three samples with moderate AD plaques and three samples with severeAD plaques). These steps eliminate DARPins reactive to antigens presentin normal or AD sera samples. Next, one round of PP was performedagainst PD brain tissue (same mixture as above) and carried out theglycine elution procedures. These phages were then added to micacontaining PD sera (from the same nine individuals that were obtainedthe brain samples). After elution one clone was acquired. DNA sequencingrevealed that this clone had a complete protein sequence with no errors.The sequence of this clone was different from the one ALS clone reactivewith both the ALS TDP-43 protein and tissue. The phages were collectedthat bound only to the PD tissue as backup.

Potential DARPins that are reactive to both AD brain tissue and serasamples are also of interest. To isolate such clones the startingmaterial was the phages that remained after the NP against FTD tissue(since the DARPins that would bind AD tissue were still present). FIG. 4highlights the portion of the schematic that summarizes the ADbiopanning procedures. It is important to note that the DARPins reactivewith PD tissue were still present since these binders were not removeduntil future NP steps. Therefore, eight rounds of NP against PD tissuewere completed using immunotubes. Next, two rounds of NP against healthyhuman sera and two rounds of NP against PD sera (using mica) werecarried out. These phages were then used in one round of PP against ADhuman brain tissue followed by glycine elution. The eluted phages wereadded to AD human sea samples. After the incubation step the boundphages were eluted. Several DARPin clones were eluted from this 2-stepPP procedure. DNA sequencing results of some of the clones revealed sixclones with complete sequences (no errors). These six clones weredifferent from the one ALS and one PD clones above. The DARPins thatwere reactive only to AD tissue were also kept.

EXAMPLE 4 Use of Serum Protein Variant Biomarkers to Assess Risk of ADfollowing TBI

Generation of nanobodies with specificity for disease specific proteinvariants. Selected protein variants including various soluble oligomericprotein species are key factors in the onset and progression of manyhuman diseases. Variants of four key neuronal proteins, tau, Aβ, TDP-43and a-syn, have been implicated in the most prevalent neurodegenerativediseases including AD, PD, FTD, ALS and other tauopathies andsynucleinopathies. The protein variants are generated by stressed cellsin diseased brains, they occur early during disease progression,different variants have different toxic effects on neuronal cells, anddifferent protein variant species preferentially occur in patients withdifferent neurodegenerative diseases. Therefore the ability to detectthe presence of specific protein variants in CSF and especially bloodsamples is a very powerful tool to help early and definitive diagnosisof different types of brain damage. Toward this end, novel biopanningtechnologies were developed that combine the imaging capabilities ofAtomic Force Microscopy (AFM) with the diversity of antibody libraries.This unique biopanning combination of antibody diversity and imagingcapability has enabled the isolation of single chain antibody variabledomain fragment (scFv or nanobody) reagents to an array of morphologiesof key proteins involved in neurodegenerative diseases including tau,Aβ, TDP-43 and a-syn. Nanobodies were isolated that specificallyrecognize monomeric (Emadi, S., et al., Biochemistry, 2004. 43(10): p.2871-2878), fibrillar (Barkhordarian, H., et al., Protein Eng Des Sel,2006. 19(11): p. 497-502), and two different oligomeric a-synmorphologies (Emadi, S., et al., J Mol Biol, 2007. 368(4): p. 1132-44;Emadi, S., et al., J Biol Chem, 2009. 284(17): p. 11048-58). Inaddition, nanobodies were isolated to different regions of monomeric(Liu, R., et al., Biochemistry, 2004. 43(22): p. 6959-67; Zameer, A., etal., Biochemistry, 2006. 45(38): p. 11532-9) and fibrillar Aβ (Marcus,W. D., et al., Nanomedicine, 2008. 4(1): p. 1-7) and to three distinctnaturally occurring oligomeric Aβ morphologies (Zameer, A., et al., JMol Biol, 2008. 384(4): p. 917-28). Nanoscale methods were developed tocharacterize nanobody binding specificities again using AFM to imagewhether the nanobodies bind different target antigens (Kasturirangan,S., et al., Biotechnol Prog, 2013. 29(2): p. 463-71). Also, nanobodieswere isolated to key forms of tau. Nanobodies were generated thatselectively bind the toxic trimeric tau species, but not monomeric orfibrillar tau. Biopanning protocols were improved in order to readilyisolate nanobodies that selectively bind disease related proteinvariants directly from minimal amounts of human samples includingtissue, CSF or sera samples. Using these biopanning protocols,nanobodies were generated that selectively bind TDP-43 variants that arepresent in both human ALS and FTD brain samples but not healthy samples,and ones that are present in ALS but not FTD or healthy and FTD but notALS or healthy brain tissue. The diversity of protein variant specificnanobodies that we are able to generate indicates the powerful andunique capabilities of our panning technology.

Detection of disease related protein variants in human samples. A)Sandwich ELISA with sub-Femtomolar sensitivity. An electronic impedancebiosensor was initially utilized to detect oligomeric protein variantsin post-mortem human CSF samples using nanobodies showing that it waspossible to distinguish between cognitively normal (ND), PD and ADsamples (Sierks, M. R., et al., Integrative Biology, 2011. 3(12): p.1188-96). To facilitate quantitative analysis of multiple samples in aformat that can be utilized in most labs, a simple yet sensitivesandwich ELISA was developed to detect low concentrations of targetantigens in human samples (Williams, S., P. Schulz, and M. R. Sierks,Biotechnol Prog, 2014). In a typical sandwich ELISA, one antibody isused to capture the target antigen and a second detection antibody,often conjugated with a fluorescent marker or enzyme such as horseradishperoxidase (HRP) is used to quantify the amount of bound target. Foroligomeric protein variants, either the same nanobody can be used toboth capture and detect the target species since the oligomericaggregates have multiple binding sites on each target molecule, or anoligomeric specific nanobody can be used to capture the target and anon-morphology selective nanobody to detect the bound target. As anexample, for the capture antibody a nanobody is used that selectivelybinds oligomeric tau, and for the detection antibody a nanobody is usedthat binds all forms of tau. To amplify the detection signal forincreased sensitivity a phage displayed version of the detectionantibody similar to what was use in the biopanning studies is used. Asingle detection antibody is thus connected to a phage particle whichcontains over 2000 copies of the gp8 coat protein (FIG. 5).Biotinylation of the coat proteins enables several thousand-foldamplification of the detection signal compared to using the nanobodyalone. A streptavidin/HRP complex is used to bind the biotinylated phageand a colorimetric or chemiluminescence substrate to quantify the boundtarget. It was possible to detect antigen over a large linearconcentration range with detection limits well below femtomolarconcentrations using a chemiluminescence substrate (Williams, S., P.Schulz, and M. R. Sierks, Biotechnol Prog, 2014). Using this sandwichELISA, it was possible to readily detect the presence of oligomericprotein aggregates not only in human brain tissue homogenates, but alsoin human CSF and serum samples as well, as shown in the followingsection.

B) Morphology Specific Nanobodies to Distinguish and Stage AD usingHuman Tissue, CSF and Serum samples. i) Human Tissue. It has been shownthat morphology specific nanobodies distinguish human post-mortem ND, ADand PD brain tissue samples. The oligomeric a-syn specific nanobodiesselectively label PD tissue (Emadi, S., et al., J Mol Biol, 2007.368(4): p. 1132-44; Emadi, S., et al., J Biol Chem, 2009. 284(17): p.11048-58) and the oligomeric Aβ specific nanobodies (Zameer, A., et al.,J Mol Biol, 2008. 384(4): p. 917-28; Kasturirangan, S., et al.,Neurobiol Aging, 2012. 33(7): p. 1320-8) and oligomeric tau specificnanobodies selectively label AD tissue. To further demonstrate thepotential value of protein variant specific nanobodies in diagnosingneurodegenerative diseases, well characterized brain samples from themiddle temporal gyrus of six non-demented (ND), six AD and nineParkinson's (PD) patients were analyzed using the sandwich ELISAprotocol described above. Post-mortem human brain tissue Wwase analyzedusing two anti-oligomeric Aβ nanobodies, A4 and C6T (Zameer, A., et al.,J Mol Biol, 2008. 384(4): p. 917-28; Kasturirangan, S., et al.,Biotechnol Prog, 2013. 29(2): p. 463-71), two anti-oligomeric a-synnanobody 10H and D5 (Emadi, S., et al., J Mol Biol, 2007. 368(4): p.1132-44; Emadi, S., et al., J Biol Chem, 2009. 284(17): p. 11048-58),and an anti-oligomeric tau nanobody F9T. Samples were prepared asdescribed (Emadi, S., et al., J Mol Biol, 2007. 368(4): p. 1132-44;Emadi, S., et al., J Biol Chem, 2009. 284(17): p. 11048-58). Proteinconcentrations of each sample were normalized and equal volumes usedwhere the samples were diluted on average around 1/70 for analysis. Whenassaying oligomeric Aβ levels, both the A4 and C6T nanobodies couldreadily distinguish between AD and PD and ND samples and could stage ADas higher levels of oligomeric Aβ were observed in AD samples withmoderate plaques compared to AD samples with severe plaques (FIG. 6).What is particularly exciting about the data is that the anti-oligomericAβ nanobodies readily distinguish between ND and AD cases even insamples that have similar amyloid plaque loads. Therefore while amyloidplaque deposition, a commonly used diagnostic test for AD, does notcorrelate very well with AD disease progression, presence of specificoligomeric Aβ species correlates very well. As expected, the oligomericAβ levels decrease as the disease progresses from moderate to severeplaque loads. When the same samples were analyzed for presence ofoligomeric a-syn using the 10H and D5 anti-oligomeric a-syn nanobodies,all the PD samples reacted strongly, whereas the AD and ND samplesshowed only background levels (FIG. 7) indicating that oligomeric a-synlevels can be used to distinguish between PD and AD and ND samples.Finally, when the same samples were analyzed for the presence ofoligomer tau using the F9T nanobody again we could readily distinguishbetween AD and ND samples with oligomeric tau levels increasing withBraak stage (FIG. 8). Of particular interest, the anti-oligomeric tauantibody can distinguish between Braak stage I and II samples with andwithout the presence of plaques, showing higher oligomeric tau levels inbrain samples that also contain the presence of slight amount of amyloidplaques. These results suggest that the presence of oligomeric tau maybe a valuable early diagnostic for AD and other tauopathies and furthervalidate that morphology specific reagents can be a powerful tool todetect different neurodegenerative diseases. When the results of thepost-mortem human brain tissue sample analysis with all the nanobodiesare combined, we have a very powerful set of reagents that can not onlyreadily distinguish between different neurodegenerative diseases, butpotentially can stage these diseases as well. While the analysis ofpost-mortem brain tissue with our nanobodies is a very powerful tool,for diagnostic applications we need to be able to make similardistinctions in CSF and ideally serum samples as well for practicalapplications. Results using post-mortem AD, PD and control CSF and serumsamples are presented in the following sections.

ii) Human Post-mortem CSF and serum. Morphology specific nanobodies canrecognize disease related protein variants in postmortem human CSF andserum samples and can distinguish between diseased and cognitivelynormal samples. The nanobodies in conjunction with an electronicbiosensor could detect the presence of oligomeric Aβ and a-syn in theseCSF samples and could readily distinguish between CSF samples from agematched cognitively normal (ND), AD and PD patients (Sierks, M.R., etal., Integrative Biology, 2011. 3(12): p. 1188-96). Here, the sandwichELISA described above was used to analyze additional post-mortem CSFsamples for presence of different oligomeric protein variants using theanti-oligomeric a-syn nanobodies D5 and 10H. CSF samples were diluted1/100 for analysis. PD was readily distinguished from AD and ND samples(FIG. 9).

A set of post-mortem serum samples were also analyzed. The serum sampleswere from the same patients used for the brain tissue studies describedabove (FIGS. 6-8). The sandwich ELISA described above was used todetermine the concentrations of oligomeric variants of Aβ and a-syn inthe serum samples. Serum samples were diluted 1/100 in PBS for analysis.When testing for oligomeric Aβ species using the C6T and A4 nanobodies,the results essentially mirrored what was obtained with the brain tissuehomogenates as it was possible to readily distinguish between AD and PDand ND samples (FIG. 10). Again very impressively, it was possible tovery readily distinguish between the AD and ND serum samples even whensimilar amyloid plaque loads were present and again higher levels ofoligomeric Aβ were observed in samples with moderate plaques and lowerlevels in samples with severe plaques. Similarly, when testing serumsamples for oligomeric a-syn species using the D5 and 10H nanobodies, itwas possible to readily distinguish between PD and AD and ND samples(FIG. 11) where higher signals were obtained with samples from earlydisease stages and lower signals with later stages.

Studies were also performed to analyze serum samples for the presence ofTDP-43 variants using a nanobody we isolated that recognizes a TDP-43variant that is present in both AD and ALS brain tissue but not healthyor FTD brain tissue. The TDP-43 protein variant was present atsignificant levels in all four AD sera samples studied, but not in thehealthy serum sample (FIG. 12). Taken together, these very excitingresults indicate the possibility of detecting the presence of specificdisease related protein variants in both CSF and serum samples and thatthe levels of the different protein variants correlate very well withspecific neurodegenerative diseases and with different stages ofdisease.

The nanobodies generated against disease related protein variants canvery effectively detect the presence of the target biomarkers inpost-mortem human tissue, CSF and sera samples and readily distinguishAD, PD and ND samples. The nanobodies can also be used to distinguishante-mortem sera or plasma samples. Longitudinal plasma samples wereobtained, and plasma samples were analyzed from six different patientsincluding two controls and four cases that converted first to MCI andthen to AD during the time period of the study. The samples wereanalyzed for the presence of different oligomeric A13 variants using theA4 (Zameer, A., et al., J Mol Biol, 2008. 384(4): p. 917-28) and E1(Kasturirangan, S., et al., Neurobiol Aging, 2012. 33(7): p. 1320-8)nanobodies. The plasma samples obtained from the two control cases didnot show any reactivity at any of the time points with either nanobody,however the samples from the four cases that converted to AD showsignificant reactivity with the nanobodies at almost every time point,even in samples taken seven years prior to an initial diagnosis of MCI(FIG. 13). These results indicate the possibility for presymptomaticdiagnosis of neurodegenerative disease using blood based protein variantbiomarkers.

Detection of specific protein variants in human tissue, CSF and serumsamples both from pathologically confirmed post-mortem samples and fromlongitudinal ante-mortem samples has great promise for presymptomaticand early diagnosis of specific neurodegenerative diseases has beenshown. Also, data was obtained that demonstrates that detection ofdisease related protein variants can be a very powerful tool to helpassess neuronal damage following TBI and to determine which patients areat risk of incurring specific neurodegenerative diseases. Sera sampleswere obtained at different time points from patients incurring severeacute TBI. To get a preliminary assessment of what the protein variantfingerprints might look like following TBI, a composite TBI sample wasgenerated by combining sera samples from five different severe TBIpatients and compared the different protein variant signals obtainedwith the composite TBI sample to a composite control sample comprised oftwo aged control samples obtained from post-mortem cognitively normalpatients (ages 83 and 89). Pooled sera samples taken 6 hours, 24 hours,5 days and 10 days following injury was analyzed for the presence oftarget protein variants of tau, Aβ, a-syn and TDP-43. The samples weretested for oligomeric tau using the F9T nanobody, for oligomeric Aβusing the A4 nanobody, for oligomeric a-syn using the 10H nanobody, andfor an AD related TDP-43 variant using a nanobody that recognized TDP-43in AD and ALS brain tissue and compared the signals obtained with theTBI samples to those obtained with the post-mortem aged control NDsamples (FIG. 14). All four protein variants were present in the variousdifferent TBI time point samples, showing different time coursedependencies. Levels of oligomeric tau and Aβ were present earlyfollowing injury and peak around 5 days after injury, levels ofoligomeric a-syn also peak around 5 days, whereas TDP-43 levels werehigh immediately after injury, decrease at 24 hours and 5 days afterinjury and increase again 10 days after injury. These studies with thecomposite sample show that different toxic protein variants aregenerated in brain tissue following severe TBI and that they can bedetected in sera samples as a potential tool to assess neuronal damage.

Each of the five severe TBI cases were analyzed separately to determineif different patients had different protein variant fingerprintsindicating potential susceptibility to different neurodegenerativediseases. Analysis of the individual cases suggests three distinctlydifferent injury patterns (FIG. 15). Patients P1 and P2 have highpersistent levels of both tau and a-syn variants following injury, butvery little or no oligomeric Aβ, while patients P3 and P4 show highpersistent levels of Aβ variants, but low levels of a-syn and tau, andpatient P5 has relatively low levels of all the protein variants tested(FIG. 15).

These biomarker results from the TBI sera samples indicate that thereare distinctly different biochemical changes occurring in brain tissueof the different patients following their respective injuries, and thatthese differences may account for increased susceptibilities for variousneurodegenerative diseases following TBI. For example, patients P1 andP2 show biochemical changes that produce toxic variants of tau and a-synand these patients are likely more susceptible to neurodegenerativedisease associated with tauopathies and synucleinopathies such as PD andFTD. Patients P3 and P4 on the other hand show biochemical changes thatproduce toxic variants of Aβ and are likely more susceptible toamyloid-based diseases such as AD or cerebral amyloid angiopathy.Further analysis indicates that patient P1 still has high levels oftoxic oligomeric tau and a-syn 10 days after injury, indicating thatsignificant neuronal damage is still occurring and likely making thiscase at greater risk of subsequent PD or FTD than patient P2. Similarly,patients P3 and P4 still have high levels of toxic oligomeric Aβ 10 daysafter injury, again indicating ongoing neuronal damage and likely makingthese patients at greater risk for AD.

Thus, it has been shown that it is possible to readily distinguishdifferent neurodegenerative diseases using sera samples from post-mortemAD and PD patients, that it is possible to readily distinguish AD fromhealthy ante-mortem sera samples even many years prior to initialdiagnosis of MCI, and that we can readily distinguish not only severeacute TBI sera samples from control samples, and also to distinguishindividual TBI cases for the presence of biomarkers indicative ofspecific neurodegenerative diseases.

EXAMPLE 5 Protein Variants Profile in Traumatic Brain Injury

Following head injury, detection of any resulting damage to the brainand the magnitude of the trauma would be advantageous for proposedtreatment strategies. The degree of damage in traumatic brain injury(TBI) can range from mild to severe and the resulting ramifications canbe immediate or gradual. Exposure of such damage via blood orcerebrospinal fluid (CSF) based biomarker detection would provide asimplified diagnostic approach and more importantly, allude to potentialimpending neurological dysfunctions including neurodegenerativedisorders like Alzheimer's, Parkinson's or Amyotrophic Lateral Sclerosis(ALS). Conformations of oligomeric beta-amyloid and tau have beenimplicated in Alzheimer's, oligomeric alpha-synuclein in Parkinson's andprotein variants of TDP-43 in ALS. The potential association between TBIand neurological deficits suggests the emergence of these proteinvariants post injury and their plausible role as indicators of trauma tothe brain. We previously isolated the single-chain variable fragments(scFvs) 3A, 3C and 8D for reactivity with variants of TDP-43, A4 and C6Tfor specificity with different conformations of oligomeric beta-amyloid,10H and D5 for recognition of certain oligomeric alpha-synucleinvariants and D11C for selection of oligomeric tau. Studies examining thebinding specificity of all eight scFvs have been published. Of late weassessed the reactivity of these scFvs with longitudinal sera and CSFsamples acquired from severe TBI patients and the details of ourfindings are presented below.

3A (SEQ ID NO: 32) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAGAGTGGGTCTCAACTATTAATACTGCTGGTAATGGTACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTACTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATTCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCTGGTGATAGTCCTGCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ 3C (SEQ ID NO: 33) 5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATTATAATTCTCCTTATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ 8D(SEQ ID NO: 34) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATAATAGTGGTACTTCTACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTAATTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCA$AAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATGCTGCTGATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ A4 (SEQ ID NO: 35)Ttgttattactcgcggcccagccggccatggccgaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatcccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagcgattcagcatactggtgcgccgacaacttacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagcgtttccgccgtttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcggtggcgggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctattctgcatcctctttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagcgggagactgggcctnnnngttcggncaanggancaangtggaaatcaaacgggcggccgcacatcatcatcaccatcacggggccgcanaacaaaaactcatctcanaanaggatctgaatggggccgcatanactgttgaaanttgtttancaaacnncatacnnnaaattcattt A4 (SEQ ID NO: 36)MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIQHTGAPTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAFPPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRETGPKAFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAA* C6T (SEQ ID NO: 37)ccatggcccaggtacagctgcaggagtcgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtgtattactgtgcgaagagctatggttcagttaaaataagctgctttgactactggggccagagcaccctggtcaccgtctcctcaggtggaggcggttcaggcggaggtggctctggcggtggcggatcggaaattgtgctgacgcagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccagccagagtgttctttacaactccaacaataagaactacttagcttggtaccagcagaaaccaggacagtctcctgagttgctcatttactgggcatcaacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagaattcactcttaccatcagcagcctgcaggctgaggatgtggcagtttattactgtcagcaattttatagtactcctccgacttttggccaggggaccaagctggagatcaaacgtgcggccgcacatcatcatcaccatcacggggccgcagaacaaaaactcatctcagaagaggatc C6T(SEQ ID NO: 38)MAQVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSYGSVKISCFDYWGQSTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQSPELLIYWASTRESGVPDRFSGSGSGTEFTLTISSLQAEDVAVYYCQQFYSTPPTFGQGTKLEIKRAAAHHHHHHGAAEQKLISEEDLNGAA* 10H (SEQ ID NO: 39)Ccatggccgaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaaatattagtagtgcagggaaggggctggagtgggtctcaagtattgatgattctggtgcttctacatattacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagattctgcttcttttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcggtggcgggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatactgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagtctgctgctagtccttctacgttcggccaagggaccaaggtggaaatcaaacgggcggccgcacatcaccatcaccatcacggggccgcagaacaaaaactcntctcagaagnggatcnnaangggnccg 10H (SEQ ID NO: 40)MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNISSAGKGLEWVSSIDDSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAASPSTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAA* D5 (SEQ ID NO: 41)ccatggccgaggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcatcgattggtcagaagggtggtggtacacagtacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaacattttgagaattttgactactggggccagggaaccctggtcaccgtctcgagcggtggaggcggttcaggcggaggtggcagcggcggtggcgggtcgacggacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcccatttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagacgcgtaggccgccttctacgttcggccaagggaccaaggtggaaatcaaacgggcggccgcacatcatcatcaccatcacggggccgcagaacaaaaactcatctcagaagagaatcactagtgcggccgcctgcaggtcgaccata D5(SEQ ID NO: 42)MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGQKGGGTQYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHFENFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRRPPSTFGQGTKVEIKRAAAHHHHHHGAAEQKLISEEDLNGAA* D11C (SEQ ID NO: 43)GCANTTCNATTTNNNGAGACAGTCATAATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCAAGAGGTGGCGATTATGGCTCAGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGAATTTTATGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGAATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGTTGGTACCAGCAGAAGCCAGGACAGGCCCCTCTCCTTGTCATCTATGGTAAAAACATCCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACTCAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTCACTCCCGGGACAGCAGTGGTACCCATCTAAGGGTATTCGGCGGAGGGACCAAGGTCACCGTCCTAGGTGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATANACTGTTGAAAGTTGTTTANCAAANNCTCATACAGAAANTTNATTNNCTANNNTCTGGNAAGANGACAAAACTTTNNNTCGTNACGCTANNNNTNNNNNNTGTCTGTGANNGCNNCNGGCNNTGTGNTNNNNACTGNNNNNNAAANTNNNGNTNNNNG D11C (SEQ ID NO: 44)A X X F X E T V I Met K Y L L P T A A A G L L L L A A Q PA Met A Q V Q L V E S G G G L V Q P G G S L R L S C A A S G F TF S S Y A Met S W V R Q A P G K G L E W V S A I S G S G G S T YY A D S V K G R F T I S R D N S K N T L Y L Q Met N S L R A E DT A V Y Y C A R G G D Y G S G D Y W G Q G T L V T V S S G G G GS G G G G S G G G G S N F Met L T Q D P A V S V A L G Q T V R IT C Q G D S L R S Y Y A S W Y Q Q K P G Q A P L L V I Y G K N IR P S G I P D R F S G S S S G N S A S L T I T G A Q A E D E A DY Y C H S R D S S G T H L R V F G G G T K V T V L G A A A E Q KL I S E E D L N G A A X T V E S C L X X X H T E X X X X X X W XX D K T X X R X A X X X X C L Stop X X X X X X X X L X X K X X X X

Longitudinal sera samples from eight cases comprising the time points 6hours, 24 hours, 5 days and 10 days post-injury were tested with ourscFvs using our previously developed phage-based capture ELISA system(Note: not every time point was available for each case). The bindingratio for each sample is represented relative to the average binding oftwo post-mortem pathologically confirmed non-demented (ND) cases.Initially, the results are displayed as spaghetti plots to illustratethe variation in binding patterns between the cases across the four timepoints, with average binding indicated in red. To begin, assessment of3A's activity indicated an average upsurge in biomarker recognition at 6hours, a decline at 24 hours and a gradual decrease post 24 hours,although, sustaining elevated reactivity relative to the controls (FIG.16). Likewise, 3C (FIG. 17) retained uniform reactivity above thecontrols across all time points, while potential biomarkers reactivewith 8D (FIG. 18) remained stable from 6 hours to 5 days but thenintensified at day 10. In general, the cumulative TDP-43 variants beingdetected peaked at 6 hours, declined at 24 hours and maintained a slightdecreasing pattern at subsequent time points (FIG. 19). Proceeding toD11C, its recognized oligomeric tau antigen increased from 6 to 24hours, and then gradually declined while concurrently retaining activitylevels exceeding the controls (FIG. 20). The average C6T reactiveoligomeric beta-amyloid level decrease slightly from 6 to 24 hours butthen increased at day 5 and remained elevated thereafter (FIG. 21).Progressing from 6 to 24 hours, D5 identified increased quantities ofoligomeric alpha-synuclein, however, in the course of transition from 24hours to day 10 the concentrations decreased (FIG. 22). Cumulativeprotein variants levels in all eight TBI cases remained elevated inrelation to the controls, however, on average there was a steady declinefrom 6 hours to day 5, followed by an abrupt decline at day 10 (FIG.23).

In the portrayed spaghetti plots, it was apparent the presence ofoutliers influencing the average binding ratios at selected time points.To identify such outliers, box plot analysis was completed for each scFvwith elimination occurring if their presence influenced the LSD one-wayANOVA test for significance. Based on the acquired statistical results,bar graphs were plotted and any significance at p≦0.05 highlighted.Clone 3A's invariable pattern of reactivity delivered significantly morereactivity at 6 hours compared to both the 5 and 10 days post-injurytime points and the controls (FIG. 24A). Likewise, 3C produced maximumactivity at 6 hours post-trauma followed by the 10-day collection point,both of whose intensities significantly exceeded those of the controls(FIG. 24B). Interestingly, following the 6 hour time lapse, the level ofthe TDP-43 protein variant recognized by 3C decreased at 24 hours,followed by a trend reversal resulting in intensity levels approachingthe ratio acquired at 6 hours. Clone 8D maintained stable reactivityacross 6 hours, 24 hours and day 5, all of which emerged significantlyelevated relative to the controls and 10 days post-injury (FIG. 24C). Asexpected, the average cumulative TDP-43 level peaked at 6 hoursgenerating a significant difference in relation to all groups except the5 day interval (FIG. 24D). It should be mentioned that positive ratioswere acquired at the other three time points indicating the existence ofour TDP-43 variants. The pattern of reactivity for D11C (FIG. 25A), C6T(FIG. 25B) and D5 (FIG. 25C) was similar to their spaghetti plots.Cumulative TDP-43 variants, oligomeric tau, oligomeric beta-amyloid andoligomeric alpha-synuclein levels was the greatest at 6 hours, followedby 5 days, both of which were significantly different from the controls(FIG. 25D).

Line graphs were created to illustrate the average binding intensitiesof the cumulative TDP-43 variants, oligomeric tau, oligomericbeta-amyloid and oligomeric alpha-synuclein with (FIG. 26) or without(FIG. 27) the outliers for each time point. Oligomeric tau andalpha-synuclein produced paralleled patterns of activity where thelevels increased from 6 to 24 hours, and then decreased subsequently;however, the reactivity with oligomeric tau was always elevated relativeto oligomeric alpha-synuclein. The patterns originating from cumulativeTDP-43 variants and oligomeric beta-amyloid levels were interestingsince for both the levels decreased from 6 to 24 hours, however, whileTDP-43's binding intensities continued in a decreasing trend, the levelof oligomeric beta-amyloid increased, suggesting an opposingmanifestation pattern in sera. This thought-provoking occurrence willrequire confirmation in future testing with a larger longitudinal samplesize. As a summary of the selective abilities of our panel of scFvs foreach time point with all 8 TBI cases, we normalized the bindingintensities yielded by each scFv to between 0 and 1 since their rangevaried, and subsequently subtracted the mean plus one standard deviationof the controls. The results presented in FIG. 28 make evident theselection of all test samples and none of the controls. Heat mappresentation of these normalized binding ratios supported the averageTDP-43 level peaking at 6 hours, oligomeric tau's and alpha-synuclein'ssimilar binding patterns with peaking at 24 hours and oligomericbeta-amyloid exhibiting its highest reactivity at the later time points,specifically 5 and 10 days post-injury (Tables 1 and 2).

TABLE 1 Heat Map of Protein Variants in Human Sera (Outliers Present)Cumulative Oligomeric Oligomeric TDP-43 Oligomeric Alpha- Beta- VariantsTau Synuclein Amyloid Controls 0.047 0.170 0.278 0.105 6 Hrs. 0.3650.450 0.556 0.176 24 Hrs. 0.271 0.507 0.736 0.153 5 Days 0.260 0.4560.616 0.240 10 Days 0.246 0.390 0.387 0.246

TABLE 2 Heat Map of Protein Variants in Human Sera (Outliers Removed)Cumulative Oligomeric Oligomeric TDP-43 Oligomeric Alpha- Beta- VariantsTau Synuclein Amyloid Controls 0.068 0.170 0.278 0.105 6 Hrs. 0.5590.450 0.556 0.176 24 Hrs. 0.324 0.507 0.736 0.153 5 Days 0.400 0.4560.616 0.240 10 Days 0.314 0.390 0.387 0.246

CSF samples from 7 of the 8 cases were available for testing along with4 ND samples. Once again, clone 3A produced diminished binding from 6hours to 5 days post-injury, followed by a sharp upsurge in intensity atday 10 (FIG. 29). This upsurge seems to be the product of one outliersample (TBI 3). 3C's reactivity increased from 6 to 24 hours anddecreased at day 5 to somewhat similar levels as day 10 (FIG. 30). 8Dreactive TDP-43 variant decreased from 6 hours to 5 days post-trauma andthen increased again at day 10 (FIG. 31). Cumulatively, TDP-43 increasedslightly from 6 to 24 hours, decreased at day 5 and then increasedsharply at day 10 (FIG. 32). With D11C, activity increased from 6 to 24hours, surged from 24 hours to 5 days followed by a sharp decreasedthereafter (FIG. 33). This surge again seems to be the result of oneoutlier (TBI 6). A4's reactivity increased from 6 to 24 hours, decreasedat day 5 and then increased again at day 10 (FIG. 34). C6T produceddecreased binding from 6 hours to day 5 and then increased at day 10(FIG. 35). For both 10H (FIG. 36) and D5 (FIG. 37), reactivity with thesamples increased from 6 to 24 hours, then decreased at day 5 followedby an increased at day 10. Overall, cumulative protein variants levelsincreased across time with a slight decrease at day 5 (FIG. 38).

Similar to above, we excluded the outliers that influenced statisticalsignificance. 3A produced it highest reactivity at the 6 hour time point(similar to sera) resulting in statistical significance compared to thecontrols (FIG. 39A). At the remaining three time points, enhancedbinding relative to the controls were observed and particularlysignificant for the 24 hour and 5 day intervals. 3C (FIG. 39B) and 8D(FIG. 39C) both produced heightened average reactivity at all timepoints relative to the controls. One interesting observation is thehighest reactivity in CSF with 3C was at the 24 hour interval, a timepoint that produced the lowest reactivity with sera. As expected, thecumulative TDP-43 binding generated maximum reactivity at the 6 hourinterval (FIG. 39D). Additionally, both the 6 and 24 hour collectionpoints were significantly different from the controls (FIG. 39D). ForD11C, peak activity was observed at 24 hours, a time point that wassignificantly different from the other groups (FIG. 40A). A4 (FIG. 40B)displayed positive reactivity with every time point compared to thecontrols, while C6T (FIG. 40C) was mainly selective of the 6 hour and 10day groups. Both 10H (FIG. 41 A) and D5 (FIG. 41B) exhibited activitywith every time point; however, D5's reactivity was significantlydifferent from the controls at each interval. Cumulatively, with allscFvs there was more reactivity with all four time points relative tothe controls with day 5 being significantly different (FIG. 41C).

Similarly, line graphs were presented to demonstrate the cumulativebinding for each protein with (FIG. 42) or without (FIG. 43) outliers.With removal of the outliers, the trend produced with cumulative TDP-43,oligomeric tau, oligomeric beta-amyloid and oligomeric alpha-synucleinwas virtually consistent, although, at the 24 hour time-point oligomerictau level was particularly intense. Following normalization of thebinding ratios for each scFv to between 0 and 1, the cumulative proteinvariants levels for most TBI time points illustrated intensitieselevated relative to the 4 control cases (FIG. 44). An interestingobservation between FIGS. 13 and 29 is that at time points wherecumulative binding was lower in the CSF for the different cases it waselevated in the sera and vice versa. These results suggest that betweenboth mediums we can identify every time point for each TBI case. In ourheat map presentation with (Table 3) or without (Table 4) outliers,removal of the outliers produced a pattern where the TDP-43 scFvs againproduced maximum reactivity at 6 hours, oligomeric tau at 6 hours,oligomeric alpha-synuclein once again at 24 hours and oligomericbeta-amyloid at 10 days.

TABLE 3 Heat Map of Protein Variants in Human CSF (Outliers Present)Cumulative Oligomeric Oligomeric TDP-43 Oligomeric Alpha- Beta- VariantsTau Synuclein Amyloid Controls 0.194 0.013 0.219 0.432 6 Hrs. 0.3390.025 0.563 0.586 24 Hrs. 0.359 0.091 0.654 0.572 5 Days 0.277 0.1640.542 0.445 10 Days 0.373 0.021 0.619 0.588

TABLE 4 Heat Map of Protein Variants in Human CSF (Outliers Removed)Cumulative Oligomeric Oligomeric TDP-43 Oligomeric Alpha- Beta- VariantsTau Synuclein Amyloid Controls 0.209 0.162 0.219 0.432 6 Hrs. 0.4830.438 0.563 0.586 24 Hrs. 0.468 0.391 0.654 0.572 5 Days 0.397 0.2270.542 0.445 10 Days 0.386 0.348 0.619 0.588

As stated earlier there was commonality between the tested sera and CSFcases, so the resulting data was reorganized to illustrate the changingprotein variants intensities between the two mediums (outliers wereremoved for these analyses). 3A's reactivity with sera decreased from 6to 24 hours, whereas the reverse occurred with CSF (FIGS. 45A, B). 24post-injury, both the sera and CSF levels decreased across time,although swifter with sera. 3C displayed mostly a decreasing bindingpattern with CSF, while for sera the reactivity at 6 hours was thegreatest followed by an abrupt decline at 24 hours and then an intenseincreased almost to the same level as at 6 hours (FIG. 45C). Based onLSD post-hoc ANOVA analysis, this produced statistically significantdifferences for both the 6 hour and 10 day time points in relation tothe 24 hour, 5 day and 10 day CSF intervals (FIG. 45D). This subgroupfurther emphasized the opposing pattern between CSF and sera as evidentby 3C's reactivity commencing 24 hours to 10 days post-injury. CSF'sreactivity with 8D generated a robust decrease in the reactivity from 6to 24 hours, whereas the decline was less intense with sera (FIG. 46A).The CSF levels continued on its declining path until day 5, whilst thereverse occurred with sera. Following day 5, there was a reversal in thebinding trend with CSF (the binding intensity increased) and remarkablythe trend with sera also inverted producing decreasing levels. 8D'sreactivity further alluded to this potential antagonistic trend betweensera and CSF. As expected the reactivity with CSF at 6 hours wassignificantly different from the CSF samples acquired at days 5 and 10and sera from day 10 (FIG. 46B). Remarkably, at day 5 where the bindingdifference between CSF and sera was at its maximum, this contrast becamesignificant. With D11C, both the CSF and sera displayed parallelpatterns of reactivity with 24 hours exhibiting maximum intensity (FIG.46C). Interestingly, the level of oligomeric tau recognized by D11C wassignificantly elevated in CSF at 24 hours compared to all the other timepoints for both CSF and sera (FIG. 46D). Proceeding to C6T, from 6 hourto 24 hours post injury, both CSF and sera generated decreasing signals(FIG. 47A). However, after 24 hours oligomeric beta-amyloid levelsincreased substantially in the sera, while CSF levels remained depressed(FIGS. 47A, B). Oligomeric beta-amyloid reactive with A4 was elevated inthe CSF at 6 and 24 hours, while there is a sharp decrease in seralevels in this time range (FIG. 47C). Actually this decrease wassignificantly different from sera samples acquired at 6 hours, 5 day and10 day time points and CSF samples collected at 6 hours and 24 hourspost-injury (FIG. 47D). Another example of this opposing trend wasfollowing 24 hours the biomarker recognized by A4 increased in sera anddecreased in CSF. With D5, reactivity with sera increase from 6 to 24hours, while in CSF there is a slight decrease (FIG. 48A). D5'sreactivity with both mediums decreased from 24 hours to 5 days andcontinued until day 10 for sera while the trend reversed for CSF. Thebinding intensity across all time points were elevated with CSF comparedto sera further providing statistically significant differences betweensera collected on day 10 and all CSF time points and the sera acquiredon day 5 relative to both the 6 and 24 hour time points with CSF (FIG.48B). Lastly, 10H's chief reaction was with sera acquired after 24hours, while continuous reactivity with CSF was detected at all timepoints (FIG. 48C). At day 10, where 10H's reactivity with CSF increased,its reactivity with sera decreased and to a significant degree (FIG.48D).

The results presented here illustrated upregulation of the proteinvariants recognized by our panel of scFvs in longitudinal sera and CSFsamples from severe TBI cases. Selection was of every time point withsera and/or CSF and little to no binding with the controls cases. Thisdistinction exemplifies the potential biomarker role of our scFvs inTBI. Diagnostic screening with our entire panel may be essential toidentify as many upregulated protein variants post-injury sincevariations can occur between individuals. This personalized screeningapproach may also prove beneficial for designing therapeutic strategies.Due to the potential neurotoxic role of these recognized proteinvariants in specific neurodegenerative disorders, their existence in TBInot only renders them likely biomarkers of trauma but also prospectivetargets to remedy the damage. The results reported in this study pointto the prospective role of our scFvs as indicators of brain trauma,potentially utilizing sera for a less painful diagnostic process.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the invention to be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

What is claimed is:
 1. A designed ankyrin repeat protein (DARPin)comprising (a) an N-Terminal Capping ankyrin repeat (AR) encoded by (SEQID NO:30), (b) a C-Terminal Capping AR encoded by (SEQ ID NO:31), and(c) three to six AR modules of about 30 to 35 amino acids, wherein eachAR module binds with a target.
 2. The DARPin of claim 1, wherein all ofthe AR modules are the same.
 3. The DARPin of claim 1, wherein one ormore of the AR modules differ.
 4. The DARPin of claim 1, wherein an ARmodule binds with TDP-43, tau, abeta or alpha-synuclein.
 5. A nucleicacid encoding the DARPin of claim
 1. 6. A vector comprising the nucleicacid of claim
 5. 7. The vector of claim 6, wherein the vector is a pIT2vector.
 8. The vector of claim 7, wherein the pIT2 vector lacks a BsaIrestriction site.
 9. The vector of claim 7, wherein the vector lacks aPelB signal and comprises a DsbA signal.
 10. A phage comprising thevector of claim
 6. 11. A method for determining risk of traumatic braininjury (TBI), assessment of the amount of neuronal damage, and/orsusceptibility to neurodegenerative disease in a subject, comprising thesteps of: (A) providing samples obtained from a subject at two or moretimes post-injury; (B) assessing levels of toxic variants of TDP-43,tau, abeta and/or alpha-synuclein in the sample by detecting toxicvariants of TDP-43, tau, abeta and/or alpha-synuclein protein levels inthe samples; (C) comparing the toxic variants of TDP-43, tau, abetaand/or alpha-synuclein protein levels in the sample at each time pointwith the toxic variant of TDP-43, tau, abeta and/or alpha-synucleinprotein levels in a normal control; and (D) determining whether thesubject has a risk of TBI in accordance with the result of step (C);wherein a subject having elevated toxic variants of TDP-43 tau, abetaand/or alpha-synuclein protein has a high risk of TBI.
 12. The method ofclaim 11, wherein a sample is obtained from the subject within 6 hourspost-injury.
 13. The method of claim 11, wherein a sample is obtainedfrom the subject about 12 to 36 hours post-injury.
 14. The method ofclaim 11, wherein a sample is obtained from the subject about 5 to 10days post injury.
 15. The method of claim 11, wherein a sample isobtained from the subject about 2 to 4 weeks days post injury.
 16. Themethod of claim 11, wherein the samples and the normal control are bloodproduct samples or cerebrospinal fluid (CSF) samples.
 17. The method ofclaim 16, wherein the blood product is serum.
 18. The method of claim11, wherein the detecting in step (B) is by means of a ligand specificfor the protein.
 19. The method of claim 18, wherein the ligand is anantibody.
 20. The method of claim 18, wherein the ligand is a designedankyrin repeat protein (DARPin).
 21. The method of claim 11, whereinprotein levels are detected by means of ELISA.
 22. The method of claim20, wherein the DARPin is encoded by a sequence having at least 90%sequence identity of any one of SEQ ID NO: 7-28.
 23. The method of claim20, wherein the DARPin is encoded by a sequence having 100% sequenceidentity of any one of SEQ ID NO: 7-28.
 24. A method for measuring thepresence of a biomarker in a human sample from a patient havingtraumatic brain injury (TBI), the improvement comprising measuring thelevels of toxic variants of TDP-43, tau, abeta and/or alpha-synuclein inthe sample for use in predicting the amount of neuronal damage, and/orsusceptibility to neurodegenerative disease in a subject.